**Meet the editor**

Dr Deniz Ekinci obtained a BSc in Chemistry in 2004, an MSC in Biochemistry in 2006 and a PhD in Biochemistry in 2009 from Atatürk University, Turkey. He studied at Stetson University, USA, in 2007-2008 and at the Max Planck Institute of Molecular Cell Biology and Genetics, Germany, in 2009-2010. Dr Ekinci currently works as an Assistant Professor of Biochemistry in the Faculty of

Agriculture, and is the Head of the Enzyme and Microbial Biotechnology Division, Ondokuz Mayıs University, Turkey. He is a member of Turkish Biochemical Society, American Chemical Society and German Genetics society. Dr Ekinci published over fifty scientific papers, reviews and book chapters and presented several conferences to scientists. He has received numerous publication awards from several scientific councils. Dr Ekinci serves as the Editor in Chief of three international books and is involved in the Editorial Board of several international journals.

Contents

**Preface IX**

Chapter 1 **Protein Purification 3**  Nison Sattayasai

Chapter 4 **Cofactor Recycling Using**

Takao Sato

**Part 1 Protein Purification & Enzymology 1**

Li Qin Zhang and Shui Qing Ye

**a Thermostable NADH Oxidase 63**  Jun-ichiro Hirano, Hiromichi Ohta, Shosuke Yoshida and Kenji Miyamoto

**to Elucidate Folding Processes 83**  Masanari Matsuoka, Michirou Kabata, Yosuke Kawai and Takeshi Kikuchi

Chapter 6 **Functional Implication Guided by Structure-Based Study on** *Catalase* **–** *Peroxidase* **(KatG) from**

Chapter 7 **Effects of Dietary Fortification of Vitamin A and**

**Folic Acid on the Composition of Chicken Egg 129**

*Haloarcula Marismortui* **99**

**Part 2 Vitamins & Antioxidants 127**

M.J. Ogundare and S.A. Bolu

Chapter 5 **Analyses of Sequences of (β/α) Barrel Proteins Based on the Inter-Residue Average Distance Statistics**

Chapter 2 **The Problems Associated with Enzyme Purification 19**  Etienne Dako, Anne-Marie Bernier, Adjéhi Thomas Dadie and Christopher K. Jankowski

Chapter 3 **Nicotinamide Phosphoribosyltransferase Inhibitors 41**  Dan Wu, Dilyara Cheranova, Daniel P. Heruth,

### Contents

#### **Preface XIII**

	- **Part 2 Vitamins & Antioxidants 127**

X Contents

Contents VII

Chapter 18 **13C-Metabolic Flux Analysis and Metabolic Regulation 385**

Tsong-Rong Yan, Chao-Fa Lee and Hung-Che Chou

Yu Matsuoka and Kazuyuki Shimizu

Chapter 19 **QCM as Cell-Based Biosensor 411**

	- **Part 3 Biotransformation & Gene Delivery 209**

#### **Part 4 Hormones and Receptors**

	- **Part 5 Organization & Regulation 341**

VI Contents

Chapter 8 **Mitochondria and Antioxidants:**

Chapter 9 **Nitric Oxide Signaling During Senescence**

Chapter 10 **Menaquinone as Well as Ubiquinone as a Crucial**

**Part 3 Biotransformation & Gene Delivery 209**

Nagy Habib and Alkiviadis Kostakis

Chapter 13 **Role of** β**-Arrestins in Endosomal Sorting of G Protein-Coupled Receptors 253**  Rohit Malik and Adriano Marchese

Chapter 14 **Structure and Function of the Lipolysis**

**Part 5 Organization & Regulation 341**

**Stimulated Lipoprotein Receptor 267**

Chapter 15 **Ghrelin, a Gastric Hormone with Diverse Functions 293** Ziru Li, Jie Luo, Yin Li and Weizhen Zhang

Chapter 16 **Polysialylation of the Neural Cell Adhesion Molecule: Setting the Stage for Plasticity Across Scales**

Jean-Chrétien Norreel and Geneviève Rougon

**of Biological Organization 343**

Michel Ronjat and Michel De Waard

Chapter 17 **What Does Maurocalcine Tell Us About**

Christophe Stenger, Catherine Corbier and Frances T. Yen

Marie-Claude Amoureux, Annelise Viallat, Coralie Giribone, Marion Benezech, Philippe Marino, Gaelle Millet, Isabelle Boquet,

**the Process of Excitation-Contraction Coupling? 365**

**Part 4 Hormones and Receptors**

**The Active Players in Islet Oxidative Stress 139** Tatyana V. Votyakova, Rita Bottino and Massimo Trucco

**and Programmed Cell Death in Leaves 159**

Chapter 11 **Genetically Modified Baker's Yeast** *Saccharomyces cerevisiae* **in Chemical Synthesis and Biotransformations 211** Ewa Białecka-Florjańczyk and Agata Urszula Kapturowska

Chapter 12 **Animal Models for Hydrodynamic Gene Delivery 235** Michalis Katsimpoulas, Dimitris Zacharoulis,

Meena Misra, Amarendra Narayan Misra and Ranjeet Singh

**Component in the** *Escherichia coli* **Respiratory Chain 187**  Naoko Fujimoto, Tomoyuki Kosaka and Mamoru Yamada

Preface

that of cell biology and pharmacology.

regulation and organization.

Chemical biology, a scientific discipline connecting the fields of chemistry and biology, refers to the application of chemical techniques and tools, synthetic chemical compounds, to the study and manipulation of biological systems. Chemical biology utilizes chemical principles to modulate systems to either investigate the underlying biology or create new function. The research area of chemical biologists is very close to

This book aims to provide a brief overview on chemistry and biology of the biomolecules, while focusing on connection of them in chemical biology. The first section opens up this book with an overview of protein purification and enzymology by introducing some important concepts. The second section addresses properties and effects of vitamins and antioxidants. The third section deals with biotransformation and gene delivery, quite popular areas. The last two sections combine signaling,

The collection of contributions from outstanding scientists and experts from different countries presents detailed information about different topics and provides a general overview about the current issues of chemical biology. The book covers a wide range of current issues, reflecting on actual problems and demonstrating the complexity of the chemical-biological interactions. I would like to tell my gratefulness to the authors around the world for their excellent contributions to the book and hope that the book will enhance the knowledge of scientists in the complexities of chemical and biological approaches; it will stimulate both professionals and students to dedicate part of their future research in understanding

**Dr. Deniz Ekinci**

Turkey

Assistant Professor of Biochemistry

Ondokuz Mays University

relevant mechanisms and applications of chemical biology.

### Preface

Chemical biology, a scientific discipline connecting the fields of chemistry and biology, refers to the application of chemical techniques and tools, synthetic chemical compounds, to the study and manipulation of biological systems. Chemical biology utilizes chemical principles to modulate systems to either investigate the underlying biology or create new function. The research area of chemical biologists is very close to that of cell biology and pharmacology.

This book aims to provide a brief overview on chemistry and biology of the biomolecules, while focusing on connection of them in chemical biology. The first section opens up this book with an overview of protein purification and enzymology by introducing some important concepts. The second section addresses properties and effects of vitamins and antioxidants. The third section deals with biotransformation and gene delivery, quite popular areas. The last two sections combine signaling, regulation and organization.

The collection of contributions from outstanding scientists and experts from different countries presents detailed information about different topics and provides a general overview about the current issues of chemical biology. The book covers a wide range of current issues, reflecting on actual problems and demonstrating the complexity of the chemical-biological interactions. I would like to tell my gratefulness to the authors around the world for their excellent contributions to the book and hope that the book will enhance the knowledge of scientists in the complexities of chemical and biological approaches; it will stimulate both professionals and students to dedicate part of their future research in understanding relevant mechanisms and applications of chemical biology.

> **Dr. Deniz Ekinci**  Assistant Professor of Biochemistry Ondokuz Mays University Turkey

**Part 1**

**Protein Purification & Enzymology**

## **Part 1**

**Protein Purification & Enzymology**

**1**

**Protein Purification**

There are many thousand kinds of proteins with different properties and functions in a cell. To study a protein, one has to obtain the highly purified intact form of the protein. A major protein is not so difficult to be purified while a minor one may need many purification steps and high skills on the techniques. This chapter describes the principles underlying techniques for separation and purification of proteins extracted from cells and tissues. The

Generally both extraction and purification processes should be done under a cold condition, mostly at 0-4oC, except for some proteins. An ice box or another cooling system is normally needed for sample cooling. The extraction procedure varies according to the type of sample and physicochemical properties of the protein. The first step is to disrupt cells or tissues. The more gentle procedure is used the more intact protein is obtained. Most of animal cells and tissues are soft and easy to break, so gentle methods can be applied. For bacteria, fungi and most plant cells which have tough cell envelope, more vigorous procedures are required. Extraction buffer or solvent is also important. Buffer with suitable concentration, ionic strength, and pH is used for extraction of water-soluble protein. In some case, mild detergent or other appropriate dissociating substance is added to the extraction buffer. Protease inhibitors are sometimes necessary to prevent destruction of the extracted proteins by proteases. From the fact that the protein extract obtained from subcellular component has contaminants less than that obtained from whole cell and will be easier to be purified. So separation of subcellular components may be done before the protein extraction. Disruption of cells should be adjusted to get intact sub-cellular components. The required cell component is obtained by centrifugation

**2.1 Extraction of water-soluble protein from animal cells and tissues**

The proteins which are components of fragile unicellular tissues such as animal blood cells can be extracted by using hypotonic buffer solution. If the sample contains different types of cells, separation of the cell types before the extraction will make the purification easier. This osmosis-based method is also used for animal cells grown in culture media. In

**1. Introduction**

strategy involves extraction and purification.

**2. Extraction of protein**

under appropriate condition.

Nison Sattayasai *Khon Kaen University*

*Thailand*

### **Protein Purification**

Nison Sattayasai *Khon Kaen University Thailand*

#### **1. Introduction**

There are many thousand kinds of proteins with different properties and functions in a cell. To study a protein, one has to obtain the highly purified intact form of the protein. A major protein is not so difficult to be purified while a minor one may need many purification steps and high skills on the techniques. This chapter describes the principles underlying techniques for separation and purification of proteins extracted from cells and tissues. The strategy involves extraction and purification.

#### **2. Extraction of protein**

Generally both extraction and purification processes should be done under a cold condition, mostly at 0-4oC, except for some proteins. An ice box or another cooling system is normally needed for sample cooling. The extraction procedure varies according to the type of sample and physicochemical properties of the protein. The first step is to disrupt cells or tissues. The more gentle procedure is used the more intact protein is obtained. Most of animal cells and tissues are soft and easy to break, so gentle methods can be applied. For bacteria, fungi and most plant cells which have tough cell envelope, more vigorous procedures are required. Extraction buffer or solvent is also important. Buffer with suitable concentration, ionic strength, and pH is used for extraction of water-soluble protein. In some case, mild detergent or other appropriate dissociating substance is added to the extraction buffer. Protease inhibitors are sometimes necessary to prevent destruction of the extracted proteins by proteases. From the fact that the protein extract obtained from subcellular component has contaminants less than that obtained from whole cell and will be easier to be purified. So separation of subcellular components may be done before the protein extraction. Disruption of cells should be adjusted to get intact sub-cellular components. The required cell component is obtained by centrifugation under appropriate condition.

#### **2.1 Extraction of water-soluble protein from animal cells and tissues**

The proteins which are components of fragile unicellular tissues such as animal blood cells can be extracted by using hypotonic buffer solution. If the sample contains different types of cells, separation of the cell types before the extraction will make the purification easier. This osmosis-based method is also used for animal cells grown in culture media. In

Protein Purification 5

is pressurized (up to 30,000 psi) by using a hydraulic pump. Shearing force is generated when the pressurized suspension is squeezed pass a very narrow outlet into the

Grinding with or without sands, in the presence of an appropriate buffer, by using a pestle and mortar actually works well with plant sample. If the plant tissue is too strong to be ground, rapid freeze with liquid nitrogen will make the plant more fragile. The frozen plant tissue is ground before shaking in the buffer (Fido et al., 2004). Some plant cells can be

Most of lipid-soluble proteins are membrane proteins. These proteins may be called proteolipids which are extracted from the samples by using organic solvents such as a mixture of chloroform and methanol (Velours et al., 1984). An aqueous solution containing mild detergents such as Triton X-100 and CHAPS is an alternative way to dissolve the proteins from cells or organelles. Strong detergents can be used but they may cause

High expression of recombinant proteins in bacterial host cell often form insoluble aggregates which is known as inclusion bodies. This form of proteins is very difficult to be solubilized. To extract any protein from bacterial inclusion bodies, strong denaturants such as 6M guanidine-HCl or 6 to 8M urea are included in the solubilizing solution. This solution efficiently extracts the aggregated protein. However, the extracted protein is also denatured and sometimes can not be renatured (Rudolph & Lilie, 1996). The other procedure is to use a mixture of mild detergents such as a combination of Triton X-100, CHAPS and sarkosyl. This method is less efficiency but more native forms of the protein

To purify any protein, various separation techniques are used depending on physical and chemical properties of the protein. The purification process can be concluded as follows.

Step 2. Detection of the desired protein Detection by size, activity or property

atmospheric pressure (Walker, 2005).

**2.3 Extraction of water-soluble protein from plant tissue**

disrupted by the mean of nitrogen decompression.

**2.4 Extraction of lipid-soluble protein**

permanent denaturation of the proteins.

**2.5 Extraction of aggregated protein**

can be recovered (Tao et al., 2010).

**3. Purification of protein**

Step 1. Crude extract of protein

some case a mild surfactant is included in the extraction buffer. Sonication or freeze/thaw cycle or a mild mechanical agitation may be used to help disruption of the cells and dissociation of the cellular components. For multicellular soft tissue, a hand homogenizer (Fig. 1) or an electrical one with optimal size is a good choice. The tiny pestle using with a micro-centrifuge tube is also commercially available for the sample with a small volume. Equipments with stronger breaking power such as a blade blender are needed for extraction of proteins from tougher tissue such as animal muscle. Cell disruption by nitrogen decompression is another method for animal cells and some plant cells. In this method, large quantities of nitrogen are dissolved in the cells under high pressure in a vessel. When the pressure is suddenly released, the dissolved nitrogen becomes bubbles. The expanding bubbles cause rupture of the cell. Intact organelles are also obtained by this method.

Fig. 1. Homogenization of tissue by a hand homogenizer.

#### **2.2 Extraction of water-soluble protein from unicellular organisms**

This group of organisms includes bacteria, yeast, fungi and some algae. Their cell envelopes are tougher than those of animal cells. The stronger methods are needed for disruption of the cells. A simple method is to shake the suspension of cells, as well as spores, with small glass beads or the other kind of beads. If the collision with the beads is done in a blender, the method is called a bead mill. Sonication is another way to break the cells. The solution used to suspend the cells can be only an appropriate buffer, or with the addition of some enzyme and/or mild detergent. The examples of the enzymes are lysozyme, cellulase and chitinase. Nonionic and zwitterionic detergents, especially Triton X-100 and CHAPS (3-[(3 cholamidopropyl) dimethylammonio]-1-propanesulfonate), are commonly used since they are mild and have little effects on protein denaturation. To disrupt the unicellular organisms with very strong envelope, a high-pressure press apparatus may be needed such as French press, Microfluidizer processors. In these systems, the cell suspension in a suitable chamber

some case a mild surfactant is included in the extraction buffer. Sonication or freeze/thaw cycle or a mild mechanical agitation may be used to help disruption of the cells and dissociation of the cellular components. For multicellular soft tissue, a hand homogenizer (Fig. 1) or an electrical one with optimal size is a good choice. The tiny pestle using with a micro-centrifuge tube is also commercially available for the sample with a small volume. Equipments with stronger breaking power such as a blade blender are needed for extraction of proteins from tougher tissue such as animal muscle. Cell disruption by nitrogen decompression is another method for animal cells and some plant cells. In this method, large quantities of nitrogen are dissolved in the cells under high pressure in a vessel. When the pressure is suddenly released, the dissolved nitrogen becomes bubbles. The expanding bubbles cause rupture of the cell. Intact organelles are also obtained by

this method.

Fig. 1. Homogenization of tissue by a hand homogenizer.

**2.2 Extraction of water-soluble protein from unicellular organisms**

This group of organisms includes bacteria, yeast, fungi and some algae. Their cell envelopes are tougher than those of animal cells. The stronger methods are needed for disruption of the cells. A simple method is to shake the suspension of cells, as well as spores, with small glass beads or the other kind of beads. If the collision with the beads is done in a blender, the method is called a bead mill. Sonication is another way to break the cells. The solution used to suspend the cells can be only an appropriate buffer, or with the addition of some enzyme and/or mild detergent. The examples of the enzymes are lysozyme, cellulase and chitinase. Nonionic and zwitterionic detergents, especially Triton X-100 and CHAPS (3-[(3 cholamidopropyl) dimethylammonio]-1-propanesulfonate), are commonly used since they are mild and have little effects on protein denaturation. To disrupt the unicellular organisms with very strong envelope, a high-pressure press apparatus may be needed such as French press, Microfluidizer processors. In these systems, the cell suspension in a suitable chamber is pressurized (up to 30,000 psi) by using a hydraulic pump. Shearing force is generated when the pressurized suspension is squeezed pass a very narrow outlet into the atmospheric pressure (Walker, 2005).

#### **2.3 Extraction of water-soluble protein from plant tissue**

Grinding with or without sands, in the presence of an appropriate buffer, by using a pestle and mortar actually works well with plant sample. If the plant tissue is too strong to be ground, rapid freeze with liquid nitrogen will make the plant more fragile. The frozen plant tissue is ground before shaking in the buffer (Fido et al., 2004). Some plant cells can be disrupted by the mean of nitrogen decompression.

#### **2.4 Extraction of lipid-soluble protein**

Most of lipid-soluble proteins are membrane proteins. These proteins may be called proteolipids which are extracted from the samples by using organic solvents such as a mixture of chloroform and methanol (Velours et al., 1984). An aqueous solution containing mild detergents such as Triton X-100 and CHAPS is an alternative way to dissolve the proteins from cells or organelles. Strong detergents can be used but they may cause permanent denaturation of the proteins.

#### **2.5 Extraction of aggregated protein**

High expression of recombinant proteins in bacterial host cell often form insoluble aggregates which is known as inclusion bodies. This form of proteins is very difficult to be solubilized. To extract any protein from bacterial inclusion bodies, strong denaturants such as 6M guanidine-HCl or 6 to 8M urea are included in the solubilizing solution. This solution efficiently extracts the aggregated protein. However, the extracted protein is also denatured and sometimes can not be renatured (Rudolph & Lilie, 1996). The other procedure is to use a mixture of mild detergents such as a combination of Triton X-100, CHAPS and sarkosyl. This method is less efficiency but more native forms of the protein can be recovered (Tao et al., 2010).

#### **3. Purification of protein**

To purify any protein, various separation techniques are used depending on physical and chemical properties of the protein. The purification process can be concluded as follows.

Step 1. Crude extract of protein

Step 2. Detection of the desired protein Detection by size, activity or property

Protein Purification 7

protein in cell, the cell disruption should be a soft procedure to get intact organelles and the other subcellular structures. The subcellular structures including organelles are then fractionated by using differential centrifugation and/or density gradient centrifugation. In differential centrifugation, the subcellular particles are separated according to their sizes into fractions by the stepwise increase of the centrifugal force. Density gradient centrifugation is a procedure to separate particles including protein aggregates by their densities. A liquid density gradient may be pre-formed before use for the separation. Various materials can be used to make the gradients such as sucrose, Ficoll. In addition the liquid density gradient can be formed during the separation. By this way, the sample is layer on top of a suitable concentration of cesium chloride. The strong centrifugation force of ultracentrifuge causes migration of cesium chloride into the bottom of the tube. The liquid density gradient is then formed by the gradient concentration of cesium chloride (Ohlendieck, 2005). The density gradient is also spontaneously generated during

Dialysis is a widely used method. The method is simple, but time consuming because the separation depends on diffusion. The sample is placed inside a dialysis bag prepared from a tube made of semipermeable membrane. Rely on commercially available dialysis tube, only small molecules whose sizes less than 10 kDa is removed from the sample to the surrounding medium. So this technique is normally used to remove salts from the solution of protein. Dialysis is also used to concentrate protein solution. Water molecules are removed from the

Molecular filtration (or ultrafiltration) is similar to dialysis. A membrane with specific pore size is used to fractionate proteins. By using pressure or centrifugation force, only the molecules smaller than the pore pass through the membrane (Fig 2). There are various sizes of the membrane pore; the largest one can cut off the large molecule whose mass is almost 100 kDa. So proteins in the sample can be separated into various ranges of molecular masses by using the membranes with different pore sizes. The protein solution is also concentrated

inside of dialysis bag by using a hydrophilic polymer such as polyethylene glycol.

centrifugation by using Percoll (polymer-coated silica particle).

Fig. 2. Separation of proteins by using molecular filtration.

by this technique.

It is not necessary to follow all steps or all techniques in the process for every protein. Some step or some technique is not necessary for some protein. For example, myoglobin is easily observed by its red color or detected by using UV-visible spectrometric method, no need to use any sophisticated procedure. Principles of separation techniques are as followings.

#### **3.1 Separation based on solubility**

Solubility of hydrophilic protein depends on its charge and hydrogen bonding with water molecule. Net charge of a protein is zero at its isoelectric pH (pI); so the protein molecule is easy to aggregate and then precipitate. The method is called isoelectric precipitation. If the pI is not known, high concentration of some salt can precipitate protein by destroying hydrogen bond between protein and water molecule. The method is called salting out or salt precipitation. Ammonium sulphate is normally used since it has high ionic strength. Proteins in crude extract can be separated into a number of fractions by gradually addition of the salt. The precipitated proteins are redissolved and used for further steps. Combination of the two methods is used for precipitation of some protein.

#### **3.2 Separation based on size and density**

The techniques include centrifugation, dialysis and molecular filtration. A high speed centrifugation (10,000-20,000g) is normally used to remove cell debris and large organelles from crude protein extract if the desired protein is a water-soluble molecule. The supernatant is then used for further purification steps. To know the location of the desired

Separation based on solubility

Separation based on molecular size and density

Chromatographic techniques

Other separation techniques

Step 4. Detection of the desired protein Detection by size, activity or property

Step 6. Detection of the desired protein Detection by size, activity or property

It is not necessary to follow all steps or all techniques in the process for every protein. Some step or some technique is not necessary for some protein. For example, myoglobin is easily observed by its red color or detected by using UV-visible spectrometric method, no need to use any sophisticated procedure. Principles of separation techniques are as followings.

Solubility of hydrophilic protein depends on its charge and hydrogen bonding with water molecule. Net charge of a protein is zero at its isoelectric pH (pI); so the protein molecule is easy to aggregate and then precipitate. The method is called isoelectric precipitation. If the pI is not known, high concentration of some salt can precipitate protein by destroying hydrogen bond between protein and water molecule. The method is called salting out or salt precipitation. Ammonium sulphate is normally used since it has high ionic strength. Proteins in crude extract can be separated into a number of fractions by gradually addition of the salt. The precipitated proteins are redissolved and used for further steps.

The techniques include centrifugation, dialysis and molecular filtration. A high speed centrifugation (10,000-20,000g) is normally used to remove cell debris and large organelles from crude protein extract if the desired protein is a water-soluble molecule. The supernatant is then used for further purification steps. To know the location of the desired

Combination of the two methods is used for precipitation of some protein.

Step 7. Determination of the purity Electrophoresis

Step 3. Separation into fractions

Step 5. Separation for the purified protein

**3.1 Separation based on solubility**

**3.2 Separation based on size and density**

protein in cell, the cell disruption should be a soft procedure to get intact organelles and the other subcellular structures. The subcellular structures including organelles are then fractionated by using differential centrifugation and/or density gradient centrifugation. In differential centrifugation, the subcellular particles are separated according to their sizes into fractions by the stepwise increase of the centrifugal force. Density gradient centrifugation is a procedure to separate particles including protein aggregates by their densities. A liquid density gradient may be pre-formed before use for the separation. Various materials can be used to make the gradients such as sucrose, Ficoll. In addition the liquid density gradient can be formed during the separation. By this way, the sample is layer on top of a suitable concentration of cesium chloride. The strong centrifugation force of ultracentrifuge causes migration of cesium chloride into the bottom of the tube. The liquid density gradient is then formed by the gradient concentration of cesium chloride (Ohlendieck, 2005). The density gradient is also spontaneously generated during centrifugation by using Percoll (polymer-coated silica particle).

Dialysis is a widely used method. The method is simple, but time consuming because the separation depends on diffusion. The sample is placed inside a dialysis bag prepared from a tube made of semipermeable membrane. Rely on commercially available dialysis tube, only small molecules whose sizes less than 10 kDa is removed from the sample to the surrounding medium. So this technique is normally used to remove salts from the solution of protein. Dialysis is also used to concentrate protein solution. Water molecules are removed from the inside of dialysis bag by using a hydrophilic polymer such as polyethylene glycol.

Molecular filtration (or ultrafiltration) is similar to dialysis. A membrane with specific pore size is used to fractionate proteins. By using pressure or centrifugation force, only the molecules smaller than the pore pass through the membrane (Fig 2). There are various sizes of the membrane pore; the largest one can cut off the large molecule whose mass is almost 100 kDa. So proteins in the sample can be separated into various ranges of molecular masses by using the membranes with different pore sizes. The protein solution is also concentrated by this technique.

Fig. 2. Separation of proteins by using molecular filtration.

Protein Purification 9

exclusion chromatography can also be used for estimation of molecular mass of protein. A set of suitable proteins with known molecular masses are separated by the same condition as the purified protein. The fraction numbers or elution volumes are then plotted against log molecular masses of the standard proteins. The standard curve is used to estimate molecular mass of the purified protein. To obtain the good result, one have to choose the right beads since different types of beads are suitable for different molecular masses of the proteins.

Fig. 3. A model demonstrates the pores in the hydrophilic gel bead used in size-exclusion

Fig. 4. Separation of two proteins by using size-exclusion chromatography. The protein mixture is loaded on the top of the gel. Then the large molecules pass the column faster than

This chromatographic technique conducts the separation according to magnitude of net electric charge of the proteins. There are two types: anion-exchange chromatography and cation-exchange chromatography. The material packed in the column is called ion exchanger which also have two types, anion and cation exchangers. Anion exchangers possess positively charged groups while cation exchangers have negatively charged groups.

chromatography.

the small molecules.

**3.3.2 Ion-exchange chromatography**

#### **3.3 Chromatographic technique**

Chromatography is the powerful method for detection and purification of biological substances. The principle of chromatographic separation is distribution or partition of separated molecules between two immiscible phases called mobile phase and stationary phase. Chromatographic methods are classified by different criteria including physical shape of stationary phase, nature of mobile phase and/or stationary phase, mechanism of separation or the other properties of the chromatographic systems. So the methods are called depending on their popularity. For examples, paper chromatography is called according to the material used as the stationary phase; thin layer chromatography (TLC) and column chromatography are named by the physical shapes of the stationary phases. Gas-liquid chromatography has gas mobile phase and liquid stationary phase. By using mechanisms of separation, chromatography is classified as adsorption chromatography, partition chromatography, size-exclusion chromatography, ion-exchange chromatography and affinity chromatography. Column chromatography is the most popular method for purification of proteins.

The conventional column chromatography is performed under low pressure. The mobile phase flows through the stationary phase in the column by the gravity or by low-pressure pump(s). So it can be called low pressure liquid chromatography (LPLC). High performance liquid chromatography (HPLC) is an advanced version of column chromatography. High pressure pumps, high-quality materials used as stationary phase and detectors with high sensitivity are used to improve the separation and to reduce the operating time. HPLC is now very popular for detection and purification of biological molecules. The other chromatographic methods are fast protein liquid chromatography (FPLC), capillary chromatography, reversed-phase liquid chromatography etc. FPLC is similar to HPLC but its operating pressure is lower than that of HPLC. LPLC, HPLC and FPLC are widely used for purification of proteins. In these methods, proteins are normally separated according to their size, charge or binding affinity. The separated proteins are eluted from the column as peaks which can be seen by various means such as absorption spectrometry, spectrofluorimetry (Wilson, 2005).

#### **3.3.1 Size-exclusion chromatography**

The other names of this method are gel filtration chromatography and gel permeation chromatography. This chromatographic technique also separates proteins by molecular sizes. Size-exclusion chromatography is the best to conserve native structure and function of the purified protein since wide varieties of buffers can be selected to obtain the suitable condition for the protein. The stationary phase of size-exclusion chromatography normally contains porous hydrophilic gel beads (Fig. 3). The gel beads used for LPLC are made of agarose, dextran, polyacrylamide, and chemical derivatives of these substances. The beads used for HPLC and FPLC are made of stronger materials such as cross-linked dextran and polystyrene. The cross-linked polystyrene can be used for separation of hydrophobic substances in the presence of organic solvents. The principle of the technique is the diffusion of molecules into the porous cavities of the beads. The molecules larger than the pores can not enter inside the beads where as the smaller ones can do. Since the pores have many sizes, the molecules including proteins are separated according to their molecular masses. The larger molecules pass the column faster than the smaller molecules (Fig. 4). Size-

Chromatography is the powerful method for detection and purification of biological substances. The principle of chromatographic separation is distribution or partition of separated molecules between two immiscible phases called mobile phase and stationary phase. Chromatographic methods are classified by different criteria including physical shape of stationary phase, nature of mobile phase and/or stationary phase, mechanism of separation or the other properties of the chromatographic systems. So the methods are called depending on their popularity. For examples, paper chromatography is called according to the material used as the stationary phase; thin layer chromatography (TLC) and column chromatography are named by the physical shapes of the stationary phases. Gas-liquid chromatography has gas mobile phase and liquid stationary phase. By using mechanisms of separation, chromatography is classified as adsorption chromatography, partition chromatography, size-exclusion chromatography, ion-exchange chromatography and affinity chromatography. Column chromatography is the most popular method for

The conventional column chromatography is performed under low pressure. The mobile phase flows through the stationary phase in the column by the gravity or by low-pressure pump(s). So it can be called low pressure liquid chromatography (LPLC). High performance liquid chromatography (HPLC) is an advanced version of column chromatography. High pressure pumps, high-quality materials used as stationary phase and detectors with high sensitivity are used to improve the separation and to reduce the operating time. HPLC is now very popular for detection and purification of biological molecules. The other chromatographic methods are fast protein liquid chromatography (FPLC), capillary chromatography, reversed-phase liquid chromatography etc. FPLC is similar to HPLC but its operating pressure is lower than that of HPLC. LPLC, HPLC and FPLC are widely used for purification of proteins. In these methods, proteins are normally separated according to their size, charge or binding affinity. The separated proteins are eluted from the column as peaks which can be seen by various means such as absorption spectrometry,

The other names of this method are gel filtration chromatography and gel permeation chromatography. This chromatographic technique also separates proteins by molecular sizes. Size-exclusion chromatography is the best to conserve native structure and function of the purified protein since wide varieties of buffers can be selected to obtain the suitable condition for the protein. The stationary phase of size-exclusion chromatography normally contains porous hydrophilic gel beads (Fig. 3). The gel beads used for LPLC are made of agarose, dextran, polyacrylamide, and chemical derivatives of these substances. The beads used for HPLC and FPLC are made of stronger materials such as cross-linked dextran and polystyrene. The cross-linked polystyrene can be used for separation of hydrophobic substances in the presence of organic solvents. The principle of the technique is the diffusion of molecules into the porous cavities of the beads. The molecules larger than the pores can not enter inside the beads where as the smaller ones can do. Since the pores have many sizes, the molecules including proteins are separated according to their molecular masses. The larger molecules pass the column faster than the smaller molecules (Fig. 4). Size-

**3.3 Chromatographic technique**

purification of proteins.

spectrofluorimetry (Wilson, 2005).

**3.3.1 Size-exclusion chromatography**

exclusion chromatography can also be used for estimation of molecular mass of protein. A set of suitable proteins with known molecular masses are separated by the same condition as the purified protein. The fraction numbers or elution volumes are then plotted against log molecular masses of the standard proteins. The standard curve is used to estimate molecular mass of the purified protein. To obtain the good result, one have to choose the right beads since different types of beads are suitable for different molecular masses of the proteins.

Fig. 3. A model demonstrates the pores in the hydrophilic gel bead used in size-exclusion chromatography.

Fig. 4. Separation of two proteins by using size-exclusion chromatography. The protein mixture is loaded on the top of the gel. Then the large molecules pass the column faster than the small molecules.

#### **3.3.2 Ion-exchange chromatography**

This chromatographic technique conducts the separation according to magnitude of net electric charge of the proteins. There are two types: anion-exchange chromatography and cation-exchange chromatography. The material packed in the column is called ion exchanger which also have two types, anion and cation exchangers. Anion exchangers possess positively charged groups while cation exchangers have negatively charged groups.

Protein Purification 11

Sepharose for purification of immunoglobulins, mannan-agarose for purification of mannosebinding lectins. (Fig. 7). After washing off the impurities, the matrix-bound protein is eluted by change of the pH of elution buffer, or by a competing substance. Affinity chromatography is always operated in a simple way by packing the matrix in a small column. Sophisticated

Fig. 6. Principle of affinity chromatography. The ligand is immobilized on a matrix and used

Fig. 7. One-step purification of mannose-binding lectin from *Dendrobium chrysotosum*. The affinity chromatography was performed by using mannan-agarose column. The crude extract (lane 1) and the purified lectin (lane 2) were separated on SDS-PAGE and then

system is not necessary for this technique.

for purification of its partner molecule.

stained by Coomassie Brilliant Blue R250.

Anion-exchange chromatography consists of anion exchanger and used for separation of proteins containing net negative charges. In contrast cation-exchange chromatography consists of cation exchanger and used for separation of proteins containing net positive charges. There are various varieties of ion exchangers depending on the matrix materials and ionic groups. Cellulose, agarose, dextran and polystyrene are used for LPLC while polystyrene, polyethers and silica are strong enough for HPLC. The matrix is chemically modified to contain ionic groups which are weakly acidic, strong acidic, weakly basic, or strong basic. The matrix with weakly acidic group (cation exchanger) as well as that with weakly basic group (anion exchanger) is suitable for separation and purification of most proteins. Choice of the exchanger depends on the pH range in which the protein is stable and the pI value of the exchanger. Generally the pH of the system should make opposite charges between the stationary phase and the protein (Fig. 5). Elution of proteins from the column can be isocratic or gradient system. The pH and ionic strength of elution buffer is important for getting well separation of proteins.

Fig. 5. Principle of ion-exchange chromatography. The anion-exchanger binds to negativecharge proteins but positive-charge proteins. The binding strength varies according to the charges of the proteins.

#### **3.3.3 Affinity chromatography**

This is an efficient technique but the material used as stationary phase is costly. The separation of molecules is based on binding affinity between macromolecules and macromolecules, or between macromolecules and low molecular mass ligands. The binding is specific for a certain molecule or a group of molecules. For examples, monoclonal antibody binds to its antigen only while avidin can bind to any biotin-coupled proteins. In this technique, one of the partner molecules is immobilized on a matrix by using a chemical reaction (Fig. 6). The matrix can be polyacrylamide, polystyrene, cross-linked dextrans and agarose etc. There are a number of chemical substances which react to different functional groups of the immobilized molecule. The chemical reaction must not destroy the binding activity. So it is necessary to use the right reaction for the immobilization. However, there are a lot of commercially available affinity matrices for different purposes such as protein A

Anion-exchange chromatography consists of anion exchanger and used for separation of proteins containing net negative charges. In contrast cation-exchange chromatography consists of cation exchanger and used for separation of proteins containing net positive charges. There are various varieties of ion exchangers depending on the matrix materials and ionic groups. Cellulose, agarose, dextran and polystyrene are used for LPLC while polystyrene, polyethers and silica are strong enough for HPLC. The matrix is chemically modified to contain ionic groups which are weakly acidic, strong acidic, weakly basic, or strong basic. The matrix with weakly acidic group (cation exchanger) as well as that with weakly basic group (anion exchanger) is suitable for separation and purification of most proteins. Choice of the exchanger depends on the pH range in which the protein is stable and the pI value of the exchanger. Generally the pH of the system should make opposite charges between the stationary phase and the protein (Fig. 5). Elution of proteins from the column can be isocratic or gradient system. The pH and ionic strength of elution buffer is

Fig. 5. Principle of ion-exchange chromatography. The anion-exchanger binds to negativecharge proteins but positive-charge proteins. The binding strength varies according to the

This is an efficient technique but the material used as stationary phase is costly. The separation of molecules is based on binding affinity between macromolecules and macromolecules, or between macromolecules and low molecular mass ligands. The binding is specific for a certain molecule or a group of molecules. For examples, monoclonal antibody binds to its antigen only while avidin can bind to any biotin-coupled proteins. In this technique, one of the partner molecules is immobilized on a matrix by using a chemical reaction (Fig. 6). The matrix can be polyacrylamide, polystyrene, cross-linked dextrans and agarose etc. There are a number of chemical substances which react to different functional groups of the immobilized molecule. The chemical reaction must not destroy the binding activity. So it is necessary to use the right reaction for the immobilization. However, there are a lot of commercially available affinity matrices for different purposes such as protein A

important for getting well separation of proteins.

charges of the proteins.

**3.3.3 Affinity chromatography**

Sepharose for purification of immunoglobulins, mannan-agarose for purification of mannosebinding lectins. (Fig. 7). After washing off the impurities, the matrix-bound protein is eluted by change of the pH of elution buffer, or by a competing substance. Affinity chromatography is always operated in a simple way by packing the matrix in a small column. Sophisticated system is not necessary for this technique.

Fig. 6. Principle of affinity chromatography. The ligand is immobilized on a matrix and used for purification of its partner molecule.

Fig. 7. One-step purification of mannose-binding lectin from *Dendrobium chrysotosum*. The affinity chromatography was performed by using mannan-agarose column. The crude extract (lane 1) and the purified lectin (lane 2) were separated on SDS-PAGE and then stained by Coomassie Brilliant Blue R250.

Protein Purification 13

with a suitable buffer are packed in a dialysis bag. The bag is placed in the buffer between two electrodes. The protein is then electrically eluted from the gel slices. One can make a simple electroelution cell or can take any commercially available apparatus. SDS is removed from the protein by dialysis in the presence of a nonionic detergent. Only a small amount of

Most proteins have been normally purified by the above techniques. To obtained intact hydrophilic protein, the purification process mainly includes salting out and chromatographic technique(s). However some proteins can be purified by simply specific procedures. For an example, molecular filtration in combination with sodium chloride solution was successfully used for purification of glutamine synthetase (Fig. 9) (Arunchaipong et al., 2009). So the simply specific procedure may be made for purification of

Fig. 9. Purification of glutamine synthetase from chick retina by using molecular filtration in the presence of 0.7M sodium chloride. The crude extract and the purified protein were separated on SDS-PAGE and then dye-stained. Lane M, molecular mass marker; lane 1,

Actually, purification process includes many steps and is laborious. Therefore it is necessary to be certain that the desired protein is present in the crude protein extract and all purification steps. For the protein with known molecular mass or size, the most versatile method is gel electrophoresis, especially SDS-polyacrylamide gel electrophoreis. Determination of enzymatic activities or other properties such as absorption spectra are also widely used. In some case the sophisticated technique, LC-MS/MS, is performed for

It is the most widely used technique for separation, detection and determination of the purity of protein. It is sometimes used for protein purification. The technique is based on

protein is obtained by electroelution and it is possibly denatured.

some protein if the protein has the specific property.

crude protein extract; lane2, purified enzyme.

identification of the protein after separation by electrophoresis.

**4. Detection of the desired protein**

**4.1 Electrophoresis**

#### **3.3.4 Hydrophobic interaction chromatography**

This chromatographic method separates proteins on the basis of hydrophobicity, the same as reversed-phase liquid chromatography (RPLC). The stationary phases of both methods are hydrophobic ligands attached to a matrix. However, hydrophobicity and number of the ligands in hydrophobic interaction chromatography (HIC) is less than those in RPLC (Fig. 8). So HIC may be referred to as a milder form of RPLC. RPLC is mainly used for separation of peptides and low molecular mass proteins that are stable in aqueous-organic solvents. HIC is suitable for purification of proteins since it uses less extreme condition for elution of the adsorbed proteins. The hydrophobic ligands of HIC are mainly alkyl (ethyl to octyl) or phenyl or polyamide groups; the matrices are cross-linked agarose or silica. For the general procedure of HIC, the sample is applied onto the column equilibrated with a mobile phase of relatively high salt concentration. The adsorbed proteins are then eluted by a solvent of decreasing salt concentration. HIC is suitable for separation of proteins after salt precipitation and/or ion-exchange chromatography since the proteins already dissolves in the solution of high salt. Although HIC gives only moderate resolution, it opens new possibilities for purifying a number of biomolecules such as receptor proteins, membrane proteins (Wilson, 2005).

Which chromatographic procedure should be used for purification of a particular protein? Ionexchange chromatography is usually the first chromatographic technique to be done for removal of unwanted proteins since the matrix is relatively cheaper and has higher binding capacity. Size-exclusion chromatography is usually operated after that. However the operation sequence can be reversed. One-step purification of protein from crude extract can be successful by using affinity chromatography (Fig. 7). Gel filtration chromatography or ion exchange chromatography is sometimes operated in accompany with affinity chromatography. Hydrophobic interaction chromatography (HIC) is another chromatographic techniques used for purification of some proteins.

Fig. 8. Comparing the structures of materials used as stationary phases in hydrophobic interaction chromatography (A) and reversed-phase liquid chromatography (B).

#### **3.4 Other separation techniques**

There are some minor methods used for purification of protein such as electroelution of the protein separated by SDS-PAGE. The stained protein band is cut out of the gel. The gel slices

This chromatographic method separates proteins on the basis of hydrophobicity, the same as reversed-phase liquid chromatography (RPLC). The stationary phases of both methods are hydrophobic ligands attached to a matrix. However, hydrophobicity and number of the ligands in hydrophobic interaction chromatography (HIC) is less than those in RPLC (Fig. 8). So HIC may be referred to as a milder form of RPLC. RPLC is mainly used for separation of peptides and low molecular mass proteins that are stable in aqueous-organic solvents. HIC is suitable for purification of proteins since it uses less extreme condition for elution of the adsorbed proteins. The hydrophobic ligands of HIC are mainly alkyl (ethyl to octyl) or phenyl or polyamide groups; the matrices are cross-linked agarose or silica. For the general procedure of HIC, the sample is applied onto the column equilibrated with a mobile phase of relatively high salt concentration. The adsorbed proteins are then eluted by a solvent of decreasing salt concentration. HIC is suitable for separation of proteins after salt precipitation and/or ion-exchange chromatography since the proteins already dissolves in the solution of high salt. Although HIC gives only moderate resolution, it opens new possibilities for purifying a number of biomolecules such as receptor proteins, membrane

Which chromatographic procedure should be used for purification of a particular protein? Ionexchange chromatography is usually the first chromatographic technique to be done for removal of unwanted proteins since the matrix is relatively cheaper and has higher binding capacity. Size-exclusion chromatography is usually operated after that. However the operation sequence can be reversed. One-step purification of protein from crude extract can be successful by using affinity chromatography (Fig. 7). Gel filtration chromatography or ion exchange chromatography is sometimes operated in accompany with affinity chromatography. Hydrophobic interaction chromatography (HIC) is another chromatographic techniques used

Fig. 8. Comparing the structures of materials used as stationary phases in hydrophobic interaction chromatography (A) and reversed-phase liquid chromatography (B).

There are some minor methods used for purification of protein such as electroelution of the protein separated by SDS-PAGE. The stained protein band is cut out of the gel. The gel slices

**3.3.4 Hydrophobic interaction chromatography**

proteins (Wilson, 2005).

for purification of some proteins.

**3.4 Other separation techniques**

with a suitable buffer are packed in a dialysis bag. The bag is placed in the buffer between two electrodes. The protein is then electrically eluted from the gel slices. One can make a simple electroelution cell or can take any commercially available apparatus. SDS is removed from the protein by dialysis in the presence of a nonionic detergent.

Most proteins have been normally purified by the above techniques. To obtained intact hydrophilic protein, the purification process mainly includes salting out and chromatographic technique(s). However some proteins can be purified by simply specific procedures. For an example, molecular filtration in combination with sodium chloride solution was successfully used for purification of glutamine synthetase (Fig. 9) (Arunchaipong et al., 2009). So the simply specific procedure may be made for purification of some protein if the protein has the specific property.

Fig. 9. Purification of glutamine synthetase from chick retina by using molecular filtration in the presence of 0.7M sodium chloride. The crude extract and the purified protein were separated on SDS-PAGE and then dye-stained. Lane M, molecular mass marker; lane 1, crude protein extract; lane2, purified enzyme.

#### **4. Detection of the desired protein**

Actually, purification process includes many steps and is laborious. Therefore it is necessary to be certain that the desired protein is present in the crude protein extract and all purification steps. For the protein with known molecular mass or size, the most versatile method is gel electrophoresis, especially SDS-polyacrylamide gel electrophoreis. Determination of enzymatic activities or other properties such as absorption spectra are also widely used. In some case the sophisticated technique, LC-MS/MS, is performed for identification of the protein after separation by electrophoresis.

#### **4.1 Electrophoresis**

It is the most widely used technique for separation, detection and determination of the purity of protein. It is sometimes used for protein purification. The technique is based on

Protein Purification 15

proteins separated on polyacrylamide gel can be visualized by many types of staining. The staining with dye especially Coomassie Brilliant Blue R250 is most widely used since it is easy, low cost and effective. Staining with some other dyes and silver staining are also available for specific purposes. Examples are Sudan Black B for staining of proteolipids,

IEF is used for determination of isoelectric pH (pI) of protein. The technique is also useful for separation, detection and determination of the purity of protein. Proteins are separated according to their pI in a medium containing pH gradient. For analytical slap gel, the medium commonly used is polyacrylamide gel or agarose gel. The pH gradient is established by a mixture of low molecular mass organic acids and bases which is called ampholytes (Berg et al., 2002). The proteins in the mixture migrate in an electrical field depending on their charges and then standstill in the gel at the pH equal to their pI (Fig. 11). The separated proteins can be visualized by an activity staining or a dye staining. The activity stain depends on the protein property. There is a problem concerning with the dye staining of IEF gel. The ampholytes can be stained with some dyes especially Coomassie

Brilliant Blue R250. So it must be removed by appropriate solutions before staining.

Fig. 11. Principle of isoelectric focusing. A pH gradient is established between two

Each protein migrates in the gel and then stop moving at the pH equal to its pI.

electrodes by using an electric field, a mixture of ampholytes and suitable electrode buffers.

For some work, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is performed to obtain a very high resolution. The technique is a sequential separation of proteins. The protein mixture is separated by using IEF in the first dimension and then SDS-PAGE in the second dimension. High skill is needed to perform the conventional procedure. Fortunately, the commercially available apparatus are designed to make it much easier and more efficient. Since the cost of the technique is rather high, it is done

After dye staining or silver staining, the protein separated by SDS-PAGE or 2D-PAGE may be identified by using Liquid Chromatography-Mass spectrometry (LC-MS). The protein band is cut from the gel and then in-gel digestion with some protease which is mostly

Thymol for staining of glycoproteins (Hames, 1981; Holtzhauer, 2006).

**4.1.2 Isoelectric focusing**

only when necessary.

migration of charged proteins in an electric field. Electrophoresis of proteins is generally conducted in a gel medium, mostly polyacrylamide. The widely used polyacrylamide gel electrophoreses (PAGE) are sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF).

#### **4.1.1 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis**

SDS-PAGE is normally employed for estimation of purity and molecular mass of protein. This versatile method is reproducible and low-cost. For the principle, sodium dodecyl sulfate (SDS) in the system binds to most proteins in amount approximately proportional to the molecular mass of the proteins. So each protein has similar charge-to-mass ratio and migrates proportional to its molecular mass. The widely used SDS-PAGE is the Laemmli protocol, as well as its modified protocols, which consists of the discontinuous buffer system. In this buffer system, there are two parts of polyacrylamide gel, stacking gel and separating gel. There are differences in composition, concentration and pH among electrode buffer, stacking buffer and separating buffer (Fig. 10). The usefulness of the buffer stem is stacking effect which occurs in the stacking gel. The effect increases concentration of proteins in the sample which helps well

Fig. 10. Polyacrylamide gel electrophoresis. The electrophoresis set composes of the upper and lower chambers which are filled with an electrode buffer. The gel is polymerized in the space between two glass plates and then connected between the two chambers. Protein samples are load in wells at the top of the gel. Protein molecules will migrate into the gel under an electric field. For SDS-PAGE, the proteins migrate from cathode to anode.

separation of the proteins in the separating gel. SDS-PAGE can be used to estimate molecular mass of the protein by using a calibration curve between relative mobility and log molecular mass of standard protein. There are the other protocols of SDS-PAGE including urea-SDS-PAGE, Tricin-SDS-PAGE etc. The other types of polyacrylamide gel electrophoresis are also available such as acid-urea-PAGE, native-PAGE. Although SDS-PAGE is commonly used, some proteins may need specific PAGE for the detection. The

migration of charged proteins in an electric field. Electrophoresis of proteins is generally conducted in a gel medium, mostly polyacrylamide. The widely used polyacrylamide gel electrophoreses (PAGE) are sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SDS-PAGE is normally employed for estimation of purity and molecular mass of protein. This versatile method is reproducible and low-cost. For the principle, sodium dodecyl sulfate (SDS) in the system binds to most proteins in amount approximately proportional to the molecular mass of the proteins. So each protein has similar charge-to-mass ratio and migrates proportional to its molecular mass. The widely used SDS-PAGE is the Laemmli protocol, as well as its modified protocols, which consists of the discontinuous buffer system. In this buffer system, there are two parts of polyacrylamide gel, stacking gel and separating gel. There are differences in composition, concentration and pH among electrode buffer, stacking buffer and separating buffer (Fig. 10). The usefulness of the buffer stem is stacking effect which occurs in the stacking gel. The effect increases concentration of proteins in the sample which helps well

Fig. 10. Polyacrylamide gel electrophoresis. The electrophoresis set composes of the upper and lower chambers which are filled with an electrode buffer. The gel is polymerized in the space between two glass plates and then connected between the two chambers. Protein samples are load in wells at the top of the gel. Protein molecules will migrate into the gel under an electric field. For SDS-PAGE, the proteins migrate from cathode to anode.

separation of the proteins in the separating gel. SDS-PAGE can be used to estimate molecular mass of the protein by using a calibration curve between relative mobility and log molecular mass of standard protein. There are the other protocols of SDS-PAGE including urea-SDS-PAGE, Tricin-SDS-PAGE etc. The other types of polyacrylamide gel electrophoresis are also available such as acid-urea-PAGE, native-PAGE. Although SDS-PAGE is commonly used, some proteins may need specific PAGE for the detection. The

**4.1.1 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis**

(SDS-PAGE) and isoelectric focusing (IEF).

proteins separated on polyacrylamide gel can be visualized by many types of staining. The staining with dye especially Coomassie Brilliant Blue R250 is most widely used since it is easy, low cost and effective. Staining with some other dyes and silver staining are also available for specific purposes. Examples are Sudan Black B for staining of proteolipids, Thymol for staining of glycoproteins (Hames, 1981; Holtzhauer, 2006).

#### **4.1.2 Isoelectric focusing**

IEF is used for determination of isoelectric pH (pI) of protein. The technique is also useful for separation, detection and determination of the purity of protein. Proteins are separated according to their pI in a medium containing pH gradient. For analytical slap gel, the medium commonly used is polyacrylamide gel or agarose gel. The pH gradient is established by a mixture of low molecular mass organic acids and bases which is called ampholytes (Berg et al., 2002). The proteins in the mixture migrate in an electrical field depending on their charges and then standstill in the gel at the pH equal to their pI (Fig. 11). The separated proteins can be visualized by an activity staining or a dye staining. The activity stain depends on the protein property. There is a problem concerning with the dye staining of IEF gel. The ampholytes can be stained with some dyes especially Coomassie Brilliant Blue R250. So it must be removed by appropriate solutions before staining.

Fig. 11. Principle of isoelectric focusing. A pH gradient is established between two electrodes by using an electric field, a mixture of ampholytes and suitable electrode buffers. Each protein migrates in the gel and then stop moving at the pH equal to its pI.

For some work, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is performed to obtain a very high resolution. The technique is a sequential separation of proteins. The protein mixture is separated by using IEF in the first dimension and then SDS-PAGE in the second dimension. High skill is needed to perform the conventional procedure. Fortunately, the commercially available apparatus are designed to make it much easier and more efficient. Since the cost of the technique is rather high, it is done only when necessary.

After dye staining or silver staining, the protein separated by SDS-PAGE or 2D-PAGE may be identified by using Liquid Chromatography-Mass spectrometry (LC-MS). The protein band is cut from the gel and then in-gel digestion with some protease which is mostly

Protein Purification 17

Determination of protein concentration is normally required during the purification process. The protein concentration is useful for determination of specific activity. There are a number of protocols to determine protein concentration such as ultraviolet absorption, Bradford method and Lowry method. Ultraviolet absorption (at 280 nm) of protein solution is the simplest method. This method is relatively sensitive and does not destroy protein; so it is used for detection of proteins eluted from chromatographic column. However, the absorption at 280 nm can be interfered by other substances such as nucleic acids. Bradford method relies on the binding between protein and the dye Coomassie Brilliant Blue G. The protein-bound dye has maximal absorption at 595 nm. In Lowry method, the protein solution is mixed with a copper sulphate solution and the Folin reagent (a mixture of sodium tungstate, molybdate and phosphate) to produces blue purple color. The color solution is quantified by the absorbance at 660 nm (Walker, 2005). However all methods used do not give true concentration of protein since it has to used some protein to make a standard calibration curve. Bovine serum albumin (BSA) is mostly used because it is high

Protein purification involves extraction and purification. In extraction process, there are different procedures to disrupt cells or tissues as well as different extraction solvents, depending on the nature of the cells or tissues. Animal cells and tissues can be disrupted by gentle methods while unicellular organisms and plant tissues require the more vigorous procedures to break the cell envelopes. Normally the extraction solvents for most hydrophilic proteins are suitable buffers except that some detergents are included in some cases. Lipid-soluble proteins are extracted by some organic solvents, or buffers containing detergents. A number of procedures are involved in the purification process such as separation based on solubility, separation based on size and density, chromatography. Chromatographic techniques are widely used and can be operated as LPLC, HPLC or FPLC. Many steps are normally required for purification of most proteins but some proteins can be purified by only simple procedures. Detection of the desired protein should be done during the purification process and electrophoreses are widely operated. Detections by activities or

Thanks go to the Ph.D. students, Miss Suganya Kumla and Miss Patthraporn Siripipatthana

Arunchaipong, K.; Sattayasai, N.; Sattayasai, J.; Svasti, J. & Rimlumduan, T. (2009). A biotin-

coupled bifunctional enzyme exhibiting both glutamine synthetase activity and glutamate decarboxylase activity. *Current Eye Research*, Vol.34, No.10, pp.809-818,

**5. Determination of protein concentration**

purity and relatively low cost.

other properties are also used for some proteins.

**6. Conclusion**

**7. Acknowledgment**

**8. References**

for their technical assistance.

ISSN 0271-3683

trypsin. The peptide fragments are separated by HPLC and injected to a series of Mass Spectrometers. This mean of mass spectrometric analysis is called tandem mass spectrometry. The MS spectra of the peptides are used to search in the data bases through the internet. The amino acid sequences of some peptides are obtained as well as the protein(s) which has them as parts of molecule (Berg et al., 2002).

#### **4.2 Enzyme activity or the other property of protein**

Apart from electrophoresis, there are some other methods for detection of the desired protein during purification process. Various specific techniques are used depending on the property of the protein. Many proteins can be stained in gel by using their enzyme activity. Since the native conformation is important for the activity, IEF and native-PAGE are compatible with activity stains. SDS-PAGE is not suitable for activity staining of protein, excepted that the proteins can be re-natured by soaking the gel with non-ionic detergent. The enzyme activity is also determined in solution. Its activity is measured by absorption spectrometry and the activity unit can be determined. Specific activity calculated from enzyme activity and the amount of protein in milligram is normally used for determination of the purity fold during the purification process. Some enzyme activity may be detected by other techniques such as paper chromatography (Arunchaipong et al., 2009).

Many proteins are not enzymes but they may have other activities or properties. Some activities or properties are used for detection of the proteins. For examples, Anti-microbial activities can be detected by microbiological methods. Glycoproteins are pursued during purification process by using lectins. In this case, the binding activity has to be done on a membrane surface since the protein molecules can not freely move in the gel matrix and the specific binding will be interfered. The membrane method is called Western blot analysis. In this method the proteins are separated using SDS-PAGE. The separated proteins are then electrically transferred onto a membrane sheet made of nitrocellulose or polyvinylidene fluoride (PVDF) (Fig. 12). The protein bands are adsorbed on the membrane surface. The membrane is then used for binding with a lectin-linked enzyme. Only glycoprotein band(s) binds to the lectin-linked enzyme. The band(s) can be vistualized by addition of a suitable substrate. Western blot analysis is also suitable for detection of protein by specific antibody.

Fig. 12. Diagrammatic representation of electrical transfer of proteins. The transfer of protein from polyacrylamide gel onto a membrane is also called western transfer. The western transfer shown in the picture is the semi-dry procedure. After separation of proteins by using polyacrylamide gel electrophoresis, the gel and the membrane sheet are sandwiched between filter papers and electrodes. The membrane (such as nitrocellulose) and the papers are wet with a transfer buffer. The proteins in the gel are transferred to the membrane by application of an electric field.

trypsin. The peptide fragments are separated by HPLC and injected to a series of Mass Spectrometers. This mean of mass spectrometric analysis is called tandem mass spectrometry. The MS spectra of the peptides are used to search in the data bases through the internet. The amino acid sequences of some peptides are obtained as well as the

Apart from electrophoresis, there are some other methods for detection of the desired protein during purification process. Various specific techniques are used depending on the property of the protein. Many proteins can be stained in gel by using their enzyme activity. Since the native conformation is important for the activity, IEF and native-PAGE are compatible with activity stains. SDS-PAGE is not suitable for activity staining of protein, excepted that the proteins can be re-natured by soaking the gel with non-ionic detergent. The enzyme activity is also determined in solution. Its activity is measured by absorption spectrometry and the activity unit can be determined. Specific activity calculated from enzyme activity and the amount of protein in milligram is normally used for determination of the purity fold during the purification process. Some enzyme activity may be detected by

Many proteins are not enzymes but they may have other activities or properties. Some activities or properties are used for detection of the proteins. For examples, Anti-microbial activities can be detected by microbiological methods. Glycoproteins are pursued during purification process by using lectins. In this case, the binding activity has to be done on a membrane surface since the protein molecules can not freely move in the gel matrix and the specific binding will be interfered. The membrane method is called Western blot analysis. In this method the proteins are separated using SDS-PAGE. The separated proteins are then electrically transferred onto a membrane sheet made of nitrocellulose or polyvinylidene fluoride (PVDF) (Fig. 12). The protein bands are adsorbed on the membrane surface. The membrane is then used for binding with a lectin-linked enzyme. Only glycoprotein band(s) binds to the lectin-linked enzyme. The band(s) can be vistualized by addition of a suitable substrate. Western blot analysis is also suitable for

Fig. 12. Diagrammatic representation of electrical transfer of proteins. The transfer of protein from polyacrylamide gel onto a membrane is also called western transfer. The western transfer shown in the picture is the semi-dry procedure. After separation of proteins by using polyacrylamide gel electrophoresis, the gel and the membrane sheet are sandwiched between filter papers and electrodes. The membrane (such as nitrocellulose) and the papers are wet with a transfer buffer. The proteins in the gel are transferred to the membrane by

protein(s) which has them as parts of molecule (Berg et al., 2002).

other techniques such as paper chromatography (Arunchaipong et al., 2009).

**4.2 Enzyme activity or the other property of protein**

detection of protein by specific antibody.

application of an electric field.

#### **5. Determination of protein concentration**

Determination of protein concentration is normally required during the purification process. The protein concentration is useful for determination of specific activity. There are a number of protocols to determine protein concentration such as ultraviolet absorption, Bradford method and Lowry method. Ultraviolet absorption (at 280 nm) of protein solution is the simplest method. This method is relatively sensitive and does not destroy protein; so it is used for detection of proteins eluted from chromatographic column. However, the absorption at 280 nm can be interfered by other substances such as nucleic acids. Bradford method relies on the binding between protein and the dye Coomassie Brilliant Blue G. The protein-bound dye has maximal absorption at 595 nm. In Lowry method, the protein solution is mixed with a copper sulphate solution and the Folin reagent (a mixture of sodium tungstate, molybdate and phosphate) to produces blue purple color. The color solution is quantified by the absorbance at 660 nm (Walker, 2005). However all methods used do not give true concentration of protein since it has to used some protein to make a standard calibration curve. Bovine serum albumin (BSA) is mostly used because it is high purity and relatively low cost.

#### **6. Conclusion**

Protein purification involves extraction and purification. In extraction process, there are different procedures to disrupt cells or tissues as well as different extraction solvents, depending on the nature of the cells or tissues. Animal cells and tissues can be disrupted by gentle methods while unicellular organisms and plant tissues require the more vigorous procedures to break the cell envelopes. Normally the extraction solvents for most hydrophilic proteins are suitable buffers except that some detergents are included in some cases. Lipid-soluble proteins are extracted by some organic solvents, or buffers containing detergents. A number of procedures are involved in the purification process such as separation based on solubility, separation based on size and density, chromatography. Chromatographic techniques are widely used and can be operated as LPLC, HPLC or FPLC. Many steps are normally required for purification of most proteins but some proteins can be purified by only simple procedures. Detection of the desired protein should be done during the purification process and electrophoreses are widely operated. Detections by activities or other properties are also used for some proteins.

#### **7. Acknowledgment**

Thanks go to the Ph.D. students, Miss Suganya Kumla and Miss Patthraporn Siripipatthana for their technical assistance.

#### **8. References**

Arunchaipong, K.; Sattayasai, N.; Sattayasai, J.; Svasti, J. & Rimlumduan, T. (2009). A biotincoupled bifunctional enzyme exhibiting both glutamine synthetase activity and glutamate decarboxylase activity. *Current Eye Research*, Vol.34, No.10, pp.809-818, ISSN 0271-3683

**2**

*1,2Canada 3Côte d'Ivoire*

**The Problems Associated**

**with Enzyme Purification**

Etienne Dako1, Anne-Marie Bernier2,

*1University of Moncton, Moncton, NB 2University of Saint-Boniface, Winnipeg, MB 3University of Abobo-Adjamé, Abidjan*

Adjéhi Thomas Dadie3 and Christopher K. Jankowski1

This chapter aims to highlight the difficulties encountered during the purification of native cellular and membrane-bound enzymes from whole cell extracts. There are many reasons for wanting to purify enzymes, such as to fully characterize them or to mass produce them for commercial purposes. Regardless of the reason for wanting to purify an enzyme, the general extraction and purification procedures are essentially the same. However, depending on the properties of the enzyme, certain modifications of the methods must be considered regarding specific problems that can be encountered throughout the process, such as enzyme insolubility and loss of enzyme activity. Although several classic and more modern methods are available to solve these kinds of challenges, the enzyme purification

We will present the general aspects of enzyme purification, followed by a specific example of the problems we encountered and attempted to resolve in the purification of the autolysin PA49.5 from *Lactococcus lactis* subsp. *cremoris*. Autolysins are bacteriolytic enzymes that digest the peptidoglycans of bacterial cell walls (Dako et al, 2008). In several studies it was found that autolysins are linked to lipids or other membranous components during the extraction process and especially during the purification. Since they are very closely linked to the cell walls, their purification presents major challenges associated with their low concentration, their insolubility and the retention of their biological activity (Marshak et al., 1996; Brown et al., 1970)**.** Among these difficulties, their insolubility during purification remains by far the most

We can look at the process of enzyme purification as a series of steps, each one sequentially enhancing the level of purification from a crude extract. It is essential to minimize protein losses throughout the process; therefore the use of fewer steps during the purification is important since losses can occur in every step due, for example, to linking to separation matrices, insolubilities, or losses into the fringe fractions during separation procedures. We will look at the general steps in the extraction of proteins from the cells, the clarification of the homogenate, the concentration or enrichment of the extract, and finally various steps in

step remains a major challenge for any method of extraction used.

serious and examples of how we tried to resolve this problem will be presented.

**1. Introduction**

### **The Problems Associated with Enzyme Purification**

Etienne Dako1, Anne-Marie Bernier2, Adjéhi Thomas Dadie3 and Christopher K. Jankowski1 *1University of Moncton, Moncton, NB 2University of Saint-Boniface, Winnipeg, MB 3University of Abobo-Adjamé, Abidjan 1,2Canada 3Côte d'Ivoire*

#### **1. Introduction**

18 Chemical Biology

Berg, J. M.; Tymoczko, J. L. & Stryer, L. (2002). *Biochemistry* (5th ed.), pp. 79-91, ISBN 0-7167-

Fido, R. J.; Mills, E. N. C.; Rigby, N. M. & Shewry, P. R. (2004). Protein extraction from

Hames, B. D. (1981). An introduction to polyacrylamide gel electrophoresis, In: *Gel*

Ohlendieck, K. (2005). Centrifugation, In: *Principles and Techniques of Biochemistry and*

Rudolph, R. & Lilie, H. (1996). *In-vitro* folding of inclusion body proteins. *The FASEB Journal*,

Tao, H.; Liu, W.; Simmons, B. N.; Harris, H. K.; Cox, C. C. & Massiah, M. A. (2010).

Velours, J.; Esparza, M.; Hoppe, J.; Sebald, W. & Guerin, B. (1984). Amino acid sequence of a

Walker, J. M. (2005). Protein structure, purification, characterization and function analysis,

Wilson, K. (2005). Chromatographic techniques, In: *Principles and Techniques of Biochemistry*

plant tissues. In Protein Purification Protocols, *Methods in Molecular Biology*,

*Electrophoresis of Proteins, A Practical Approach*, Hames, B. D. and Rickwood, D.

*Molecular Biology* (6th ed.), Wilson, K. and Walker, J. (Eds.), pp. 103-130, ISBN 0-521-

Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-

new mitochondrially synthesized proteo-lipid of the ATP synthase of *Saccharomyces*

In: *Principles and Techniques of Biochemistry and Molecular Biology* (6th ed.), Wilson, K. and Walker, J. (Eds.), pp. 349-404, ISBN 0-521-53581-6, Cambridge University Press,

*and Molecular Biology* (6th ed.), Wilson, K. and Walker, J. (Eds.), pp. 485-550, ISBN 0-

4684-0, W.H. Freeman and Company, New York

Holtzhauer, M. (2006). *Basic Methods for the biochemical Lab*, Springer, Berlin

53581-6, Cambridge University Press, Cambridge

100, and CHAPS. *BioTechniques*, Vol.48, No.1, pp.61-64

*cerevisiae*. *The EMBO Journal*, Vol.3, No.1, pp. 207-212

521-53581-6, Cambridge University Press, Cambridge

Vol.244, pp. 21-27

Vol.10, pp. 49-56

Cambridge

(Eds.) pp. 1-86. IRL Press, Oxford

This chapter aims to highlight the difficulties encountered during the purification of native cellular and membrane-bound enzymes from whole cell extracts. There are many reasons for wanting to purify enzymes, such as to fully characterize them or to mass produce them for commercial purposes. Regardless of the reason for wanting to purify an enzyme, the general extraction and purification procedures are essentially the same. However, depending on the properties of the enzyme, certain modifications of the methods must be considered regarding specific problems that can be encountered throughout the process, such as enzyme insolubility and loss of enzyme activity. Although several classic and more modern methods are available to solve these kinds of challenges, the enzyme purification step remains a major challenge for any method of extraction used.

We will present the general aspects of enzyme purification, followed by a specific example of the problems we encountered and attempted to resolve in the purification of the autolysin PA49.5 from *Lactococcus lactis* subsp. *cremoris*. Autolysins are bacteriolytic enzymes that digest the peptidoglycans of bacterial cell walls (Dako et al, 2008). In several studies it was found that autolysins are linked to lipids or other membranous components during the extraction process and especially during the purification. Since they are very closely linked to the cell walls, their purification presents major challenges associated with their low concentration, their insolubility and the retention of their biological activity (Marshak et al., 1996; Brown et al., 1970)**.** Among these difficulties, their insolubility during purification remains by far the most serious and examples of how we tried to resolve this problem will be presented.

We can look at the process of enzyme purification as a series of steps, each one sequentially enhancing the level of purification from a crude extract. It is essential to minimize protein losses throughout the process; therefore the use of fewer steps during the purification is important since losses can occur in every step due, for example, to linking to separation matrices, insolubilities, or losses into the fringe fractions during separation procedures. We will look at the general steps in the extraction of proteins from the cells, the clarification of the homogenate, the concentration or enrichment of the extract, and finally various steps in

The Problems Associated with Enzyme Purification 21

solubilization of a protein is its ability to not only solubilize but to maintain biological activity. In general, the detergents with higher critical micelle concentrations (CMC) can more easily denature proteins than those with lower CMC (Linke, 2009; Lever et al. 1994; Garavito, 1991; Hjelmeland & Chrambach, 1984), and it must be used at concentrations

Once the cell or tissue homogenate is produced, the cell debris and other insoluble compounds must be removed by centrifugation. The optimal situation is that following a high speed centrifugation, the solubilized proteins will be in the supernatant along with other soluble compounds, and that all insoluble material will be in precipitate. Sequential or differential precipitations can be performed to progressively remove cell debris in order to rehomogenize it to enhance protein yield or to isolate specific cell organelles if the starting material is eukaryotic. Following this clarification, the cell extract is then ready to be

A process that can be considered after the clarification is the concentration of the enzyme preparation. Dialysis can be performed prior to the concentration procedure in order to remove salts from the cell extract. The clarified cell extract can therefore be dialyzed against a large volume of buffer to remove salts and the dialysate can then be lyophilized to concentrate the extract. Lyophilisation, or freeze-drying, has the advantage of protecting the protein from the chemical reactions that can damage the proteins in an aqueous solution, such as aggregation, precipitation and protease degradation (O'Fagain 2004). The protein extract can then be stored in the lyophilized state and aliquots resuspended as needed.

The most common concentration procedures are ammonium sulfate precipitation,

There are two applications of ammonium sulfate precipitation. The first one is a complete precipitation in which all proteins are precipitated by the addition of the salt. The proteins are recovered by centrifugation and resuspended in a minimal volume of water and are therefore concentrated. The residual salt must be removed by dialysis as it can interfere with protein activity or subsequent purification steps. One of the problems encountered with the dialysis of a protein extract is that the protein can precipitate onto the walls of the dialysis tubing thereby impairing the passage of particles across the membrane. This would be particularly problematic in the dialysis of a crude cell extract (Doonan, 2004a). The salts cause the precipitation of proteins by removing water from the hydrophobic patches on the protein's surface, thus rendering them insoluble (Doonan, 2004b). Another approach to the concentration of a cell extract is a fractional precipitation using a salt. Again ammonium sulfate is the most frequently used salt, because of its high solubility and the fact that the density of saturated solutions is less that of the protein. This allows the collection of proteins by centrifugation (Doonan, 2004b). The fractional precipitation is done by incrementally adding amounts of ammonium sulfate and collecting the fractions precipitated by various salt concentrations by centrifugation. Fractions can, for example, be collected at each increment of 20% concentration of salt. Each fraction can be analyzed for the enzyme in question. This will therefore concentrate the cell extract and also begin the process of purification. Depending on the solubility of the protein it may however be found in more than one fraction. The combination of these fractions will increase the yield of the enzyme

ultrafiltration and ion exchange chromatography (Ward & Swiatek, 2009).

above its CMC in order to effectively solubilize the proteins (Privé, 2007).

purified in an enrichment or concentration procedure.

however this will also reduce the level of purification.

the final purification of the enzyme. A purification strategy can be made more efficient if certain physical or chemical properties such as molecular weight, optimal pH, isoelectric point, of the enzyme are known.

To follow the efficacy of the purification of the target enzyme from a whole cell extract there must be a method of measuring its presence throughout the process to ensure the maximum yield. These assays can be an assessment of biological activity or assays based on reactions with antibodies. The antibody assays however do not necessarily indicate if the enzyme is in an active conformation. The assays based on catalytic activity do however require that the enzyme retain its active conformation, which can be problematic since many components of the extractions buffers contain compounds such as detergents that denature the tridimensional structure of the enzyme. Compounds compatible with enzyme assays must therefore be used to minimize losses of activity. The isolation of certain types of enzymes may present problems specific to those enzymes. For example, some problems most often associated with the purification of membrane proteins are that they require organic solvents or detergents for their solubilization. Also, insoluble enzymes may require the use of stronger chaotropic compounds such as lithium chloride or guanidine HCL that can in turn inactivate the enzyme. In order to be able to fully characterize the biochemical properties of a purified enzyme, it must be completely free of contaminants such as cell wall or cell membrane components, nucleic acids or other proteins. The complete removal of contaminants is, therefore, the primary objective of the purification. These contaminants can interfere with subsequent purification steps or with enzyme activity assays. The purity of an enzyme extraction can be confirmed, for example, by the presence of a single band on an electrophoretic gel (SDS-PAGE or isoelectric focusing), a single peak in an HPLC analysis or by N-terminal amino acid sequencing.

The initial step in all protein purification protocols is the release of the enzyme from the cell or tissue material, or cell extract preparation. This is a critical step in the process as it affects protein yield and biological activity of the target enzyme. This naturally requires either a mechanical or chemical lysis of the cells. Methods for homogenizing cells or tissues vary from gentle methods such as osmotic shock, detergent lysis or enzymatic digestion to more vigorous methods such as homogenization in a blender, grinding with an abrasive substance or ultrasonication. The lysis procedure selected must not damage the target enzyme and therefore the conditions must be optimized for each cell type and target enzyme in terms of the pH of the extraction buffer, the temperature and the concentration of certain components of the buffer such as detergents, salts or reducing agents for example. Reducing agents such a dithiothreitol or 2-mercaptoethanol can be used to prevent the oxidation of sulfhydryl groups that could damage the active conformation of the enzyme resulting in a loss of activity (Ward & Swiatek, 2009; Chen et al., 2010). Detergents or organic solvents, generally used to isolate hydrophobic or membrane bound enzymes, can be problematic in subsequent purification steps as well as interfere with the native conformation of the enzyme. It is essential also to consider subsequent purification steps when choosing a detergent. Certain downstream applications such as optical spectroscopy, mass spectrometry and crystallization, can be negatively affected by the presence of some detergents (Linke, 2009). There are various detergents available and these will be discussed further in the section of enzyme purification. The most important criteria for the selection of a detergent for the

the final purification of the enzyme. A purification strategy can be made more efficient if certain physical or chemical properties such as molecular weight, optimal pH, isoelectric

To follow the efficacy of the purification of the target enzyme from a whole cell extract there must be a method of measuring its presence throughout the process to ensure the maximum yield. These assays can be an assessment of biological activity or assays based on reactions with antibodies. The antibody assays however do not necessarily indicate if the enzyme is in an active conformation. The assays based on catalytic activity do however require that the enzyme retain its active conformation, which can be problematic since many components of the extractions buffers contain compounds such as detergents that denature the tridimensional structure of the enzyme. Compounds compatible with enzyme assays must therefore be used to minimize losses of activity. The isolation of certain types of enzymes may present problems specific to those enzymes. For example, some problems most often associated with the purification of membrane proteins are that they require organic solvents or detergents for their solubilization. Also, insoluble enzymes may require the use of stronger chaotropic compounds such as lithium chloride or guanidine HCL that can in turn inactivate the enzyme. In order to be able to fully characterize the biochemical properties of a purified enzyme, it must be completely free of contaminants such as cell wall or cell membrane components, nucleic acids or other proteins. The complete removal of contaminants is, therefore, the primary objective of the purification. These contaminants can interfere with subsequent purification steps or with enzyme activity assays. The purity of an enzyme extraction can be confirmed, for example, by the presence of a single band on an electrophoretic gel (SDS-PAGE or isoelectric focusing), a single peak in an HPLC analysis or

The initial step in all protein purification protocols is the release of the enzyme from the cell or tissue material, or cell extract preparation. This is a critical step in the process as it affects protein yield and biological activity of the target enzyme. This naturally requires either a mechanical or chemical lysis of the cells. Methods for homogenizing cells or tissues vary from gentle methods such as osmotic shock, detergent lysis or enzymatic digestion to more vigorous methods such as homogenization in a blender, grinding with an abrasive substance or ultrasonication. The lysis procedure selected must not damage the target enzyme and therefore the conditions must be optimized for each cell type and target enzyme in terms of the pH of the extraction buffer, the temperature and the concentration of certain components of the buffer such as detergents, salts or reducing agents for example. Reducing agents such a dithiothreitol or 2-mercaptoethanol can be used to prevent the oxidation of sulfhydryl groups that could damage the active conformation of the enzyme resulting in a loss of activity (Ward & Swiatek, 2009; Chen et al., 2010). Detergents or organic solvents, generally used to isolate hydrophobic or membrane bound enzymes, can be problematic in subsequent purification steps as well as interfere with the native conformation of the enzyme. It is essential also to consider subsequent purification steps when choosing a detergent. Certain downstream applications such as optical spectroscopy, mass spectrometry and crystallization, can be negatively affected by the presence of some detergents (Linke, 2009). There are various detergents available and these will be discussed further in the section of enzyme purification. The most important criteria for the selection of a detergent for the

point, of the enzyme are known.

by N-terminal amino acid sequencing.

solubilization of a protein is its ability to not only solubilize but to maintain biological activity. In general, the detergents with higher critical micelle concentrations (CMC) can more easily denature proteins than those with lower CMC (Linke, 2009; Lever et al. 1994; Garavito, 1991; Hjelmeland & Chrambach, 1984), and it must be used at concentrations above its CMC in order to effectively solubilize the proteins (Privé, 2007).

Once the cell or tissue homogenate is produced, the cell debris and other insoluble compounds must be removed by centrifugation. The optimal situation is that following a high speed centrifugation, the solubilized proteins will be in the supernatant along with other soluble compounds, and that all insoluble material will be in precipitate. Sequential or differential precipitations can be performed to progressively remove cell debris in order to rehomogenize it to enhance protein yield or to isolate specific cell organelles if the starting material is eukaryotic. Following this clarification, the cell extract is then ready to be purified in an enrichment or concentration procedure.

A process that can be considered after the clarification is the concentration of the enzyme preparation. Dialysis can be performed prior to the concentration procedure in order to remove salts from the cell extract. The clarified cell extract can therefore be dialyzed against a large volume of buffer to remove salts and the dialysate can then be lyophilized to concentrate the extract. Lyophilisation, or freeze-drying, has the advantage of protecting the protein from the chemical reactions that can damage the proteins in an aqueous solution, such as aggregation, precipitation and protease degradation (O'Fagain 2004). The protein extract can then be stored in the lyophilized state and aliquots resuspended as needed.

The most common concentration procedures are ammonium sulfate precipitation, ultrafiltration and ion exchange chromatography (Ward & Swiatek, 2009).

There are two applications of ammonium sulfate precipitation. The first one is a complete precipitation in which all proteins are precipitated by the addition of the salt. The proteins are recovered by centrifugation and resuspended in a minimal volume of water and are therefore concentrated. The residual salt must be removed by dialysis as it can interfere with protein activity or subsequent purification steps. One of the problems encountered with the dialysis of a protein extract is that the protein can precipitate onto the walls of the dialysis tubing thereby impairing the passage of particles across the membrane. This would be particularly problematic in the dialysis of a crude cell extract (Doonan, 2004a). The salts cause the precipitation of proteins by removing water from the hydrophobic patches on the protein's surface, thus rendering them insoluble (Doonan, 2004b). Another approach to the concentration of a cell extract is a fractional precipitation using a salt. Again ammonium sulfate is the most frequently used salt, because of its high solubility and the fact that the density of saturated solutions is less that of the protein. This allows the collection of proteins by centrifugation (Doonan, 2004b). The fractional precipitation is done by incrementally adding amounts of ammonium sulfate and collecting the fractions precipitated by various salt concentrations by centrifugation. Fractions can, for example, be collected at each increment of 20% concentration of salt. Each fraction can be analyzed for the enzyme in question. This will therefore concentrate the cell extract and also begin the process of purification. Depending on the solubility of the protein it may however be found in more than one fraction. The combination of these fractions will increase the yield of the enzyme however this will also reduce the level of purification.

The Problems Associated with Enzyme Purification 23

According to Brown (1972)**,** the problem of the solubility of enzymes during purification is linked to the presence of material other than autolysins, which absorb strongly at 260 to 280 nm. According to Shockman *et al.* (1967)**,** the treatment of the cell wall by sodium dodecyl sulfate (SDS) would resolve the problem of the insolubility of the autolysins of lactic bacteria during purification. SDS is the most widely used detergent to solubilize most otherwise insoluble proteins such as autolysins (Dolinger et al., 1989)**,** its presence may affect the tertiary structure of proteins because it is one of the most strongly denaturing detergents. It is also one of the most difficult to remove with dialysis. The renaturing of SDS extracted proteins after electrophoresis in denaturing conditions does not totally eliminate the SDS, even if it allows the activity of lytic proteins (Lortal et al., 1997) to be measured. Thus, we propose that the use of other non ionic (Triton X-100) or zwitterion (CHAPS) non-denaturing detergents will have the advantage of resolving the problem of the insolubility of autolysins whilst protecting their structure. In general, the detergents such as CHAPS and those that possess a cholanoic group such as cholate all produce very small micelles and have a critical value of micellar concentration (CMC) below 2mM. The CMC is the value above which there is the formation of micelles (Hjelmeland et Chrambach, 1984; Helenius et al., 1979)**.** These detergents can be eliminated by dialysis without affecting the protein activity (Hjelmeland et Chrambach, 1984)**.** The main aim of this study was to find a non-denaturing method to solubilize the main autolysin (PA49.5) of *L. lactis* subsp. *cremoris* ATCC 9596 (Mc5) so as to ensure its

purification from the supernatant of cellular remnants and whole cells.

hydrophobic enzymes.

this enzyme.

In several studies it was found that autolysins are linked to lipids or other membranous components during the extraction process and especially during the purification. They are therefore considered to be hydrophobic enzymes; however, it is often unclear whether or not the enzyme of interest is in fact hydrophobic. Although several classic and more modern methods are available to solve these kinds of challenges, there is still much uncertainty, and further studies are still needed to clearly resolve the purification of cell membrane

An important observation, reported by Brown et al (1970), is that autolysins are associated to teichoic acid in the cell wall of *Bacillus subtilis*. These authors propose that this acid can be eliminated by ethanol precipitation and by gel filtration on an agarose column. Their finding did not confirm this hypothesis, in fact, they found that only 5% of the techoic acid was eliminated using this method. This acid also persisted after multiple washes with ethanol or ether, and it persisted after separation in a polyacrylamide gradient density gel

After many attempts, certain autolysins were purified by electrophoresis using SDS as the detergent (*N*-acétylglucosaminidase, ac-*N*-acetylmuramidase and ac-*N*-acetylmuramyl-Lalanine amidase) (Motoyuki et al. 1995). Brown (1972) suggests that the purification of autolysines from lactic bacteria is only possible in the presence of SDS. While SDS is an excellent approach to the purification of this type of enzyme, we propose that another method must be found in order to eliminate the compounds that bind to the autolysin thereby rendering it insoluble. CHAPS, a zwiterrionic detergent, used at high temperatures was evaluated as a potentially more efficient detergent in the purification of

electrophoresis, ion exchange chromatography and gel filtration using 7M urea.

Ultrafiltration is based on the principal of separation through a semi permeable membrane filter. Many filter type units are available commercially, either applying a centrifugal or stirred cell type of separation (Bonner, 2007). The principal for all ultra centrifugal methods is the filtration through a membrane that has a selective molecular weight cut off point, therefore it will separate the extract based on the size of particles, not the charge.

Ion exchange chromatography is a non affinity absorption method that purifies proteins on the function of molecular charge, not the molecular weight. The resin in the chromatography column is either negatively or positively charged. Proteins also have a charge that will be dependent on the pH of the extract or solution. The principal of ion exchange chromatography is to pass the protein extract through the column at a pH where the protein will adhere to the oppositely charged matrix. The pH is then gradually changed during the chromatography and the proteins will be eluted when their charge is neutralized by the pH of the elution buffer. Fractions are collected from the column and analyzed for enzyme activity to identify the fraction containing the enzyme in question (Bonner, 2007).

There are no set protocols or sequences of procedures that will ensure the purification of an enzyme from a cell extract. These will vary according to the source of the enzyme and the specific properties of the enzyme.

To better understand the difficulties and techniques of extraction and purification, we decided to present a specific example, that of the lytic membrane enzyme PA49.5 so named for its molecular mass of 49.5 kDa. This enzyme was isolated from *Lactococcus lactis* subsp. *cremoris* ATCC 9596 denoted MC5. The objective of our study was to find a non-denaturing method to solubilize the autolysin in order to ensure its purification to a point that would allow its biochemical characterization. The extraction of PA49.5 from a crude cell extract supernatant, from cell wall debris and whole cells was attempted using different approaches in order to attempt to maximize solubilization of the enzyme. Extracts were further solubilized in various detergents and organic compounds and subsequently further purified by HPLC, ion exchange chromatography or ammonium sulfate precipitation.

In view of their richness in ripening enzymes (Dako et al. 2008; Dako et al. 1995), lactococci play an essential role in the production of cheese. These microorganisms also contain lytic enzymes known as autolysins because of their capacity to degrade the membranes of the bacteria that produce them. A few examples of the autolysins are: *N-*acetylmuramidases, Nacetylglucosaminidases, N-acetylmuramyl-L-alanine amidase and peptidases (Dako et al., 2003; Valence et Lortal., 1995; Shockman et Höltje, 1994; Kawagishi et al., 1980). These autolysins have an effect on specific areas in the ß-1,4 bonds between the Nacetylglucosamine and the N-acetylmuramic acid (N-acetylglucosaminidases, Nacetylmuramyl-L-alanine amidases) or between N-acetylmuramic and N-acetylglucosamine (N-acetylmuramidases). Since they are very closely linked to the cell walls of these bacteria, the isolation of autolysins presents major difficulties associated with their low concentration, their insolubility and the retention of their activity (Marshak et al., 1996; Brown et al., 1970)**.** Among these difficulties, the insolubility of autolysins during purification remains by far the most serious problem. This is due to the presence of teichoic (Lortal et al., 1997) and lipoteichoic (Fisher et al., 1980; Höltje & Tomasz, 1975) acids.

Ultrafiltration is based on the principal of separation through a semi permeable membrane filter. Many filter type units are available commercially, either applying a centrifugal or stirred cell type of separation (Bonner, 2007). The principal for all ultra centrifugal methods is the filtration through a membrane that has a selective molecular weight cut off point, therefore it will separate the extract based on the size of particles, not

Ion exchange chromatography is a non affinity absorption method that purifies proteins on the function of molecular charge, not the molecular weight. The resin in the chromatography column is either negatively or positively charged. Proteins also have a charge that will be dependent on the pH of the extract or solution. The principal of ion exchange chromatography is to pass the protein extract through the column at a pH where the protein will adhere to the oppositely charged matrix. The pH is then gradually changed during the chromatography and the proteins will be eluted when their charge is neutralized by the pH of the elution buffer. Fractions are collected from the column and analyzed for enzyme activity to identify the fraction containing the enzyme in question

There are no set protocols or sequences of procedures that will ensure the purification of an enzyme from a cell extract. These will vary according to the source of the enzyme and the

To better understand the difficulties and techniques of extraction and purification, we decided to present a specific example, that of the lytic membrane enzyme PA49.5 so named for its molecular mass of 49.5 kDa. This enzyme was isolated from *Lactococcus lactis* subsp. *cremoris* ATCC 9596 denoted MC5. The objective of our study was to find a non-denaturing method to solubilize the autolysin in order to ensure its purification to a point that would allow its biochemical characterization. The extraction of PA49.5 from a crude cell extract supernatant, from cell wall debris and whole cells was attempted using different approaches in order to attempt to maximize solubilization of the enzyme. Extracts were further solubilized in various detergents and organic compounds and subsequently further purified

In view of their richness in ripening enzymes (Dako et al. 2008; Dako et al. 1995), lactococci play an essential role in the production of cheese. These microorganisms also contain lytic enzymes known as autolysins because of their capacity to degrade the membranes of the bacteria that produce them. A few examples of the autolysins are: *N-*acetylmuramidases, Nacetylglucosaminidases, N-acetylmuramyl-L-alanine amidase and peptidases (Dako et al., 2003; Valence et Lortal., 1995; Shockman et Höltje, 1994; Kawagishi et al., 1980). These autolysins have an effect on specific areas in the ß-1,4 bonds between the Nacetylglucosamine and the N-acetylmuramic acid (N-acetylglucosaminidases, Nacetylmuramyl-L-alanine amidases) or between N-acetylmuramic and N-acetylglucosamine (N-acetylmuramidases). Since they are very closely linked to the cell walls of these bacteria, the isolation of autolysins presents major difficulties associated with their low concentration, their insolubility and the retention of their activity (Marshak et al., 1996; Brown et al., 1970)**.** Among these difficulties, the insolubility of autolysins during purification remains by far the most serious problem. This is due to the presence of teichoic (Lortal et al., 1997) and lipoteichoic (Fisher et al., 1980; Höltje & Tomasz, 1975) acids.

by HPLC, ion exchange chromatography or ammonium sulfate precipitation.

the charge.

(Bonner, 2007).

specific properties of the enzyme.

According to Brown (1972)**,** the problem of the solubility of enzymes during purification is linked to the presence of material other than autolysins, which absorb strongly at 260 to 280 nm. According to Shockman *et al.* (1967)**,** the treatment of the cell wall by sodium dodecyl sulfate (SDS) would resolve the problem of the insolubility of the autolysins of lactic bacteria during purification. SDS is the most widely used detergent to solubilize most otherwise insoluble proteins such as autolysins (Dolinger et al., 1989)**,** its presence may affect the tertiary structure of proteins because it is one of the most strongly denaturing detergents. It is also one of the most difficult to remove with dialysis. The renaturing of SDS extracted proteins after electrophoresis in denaturing conditions does not totally eliminate the SDS, even if it allows the activity of lytic proteins (Lortal et al., 1997) to be measured. Thus, we propose that the use of other non ionic (Triton X-100) or zwitterion (CHAPS) non-denaturing detergents will have the advantage of resolving the problem of the insolubility of autolysins whilst protecting their structure. In general, the detergents such as CHAPS and those that possess a cholanoic group such as cholate all produce very small micelles and have a critical value of micellar concentration (CMC) below 2mM. The CMC is the value above which there is the formation of micelles (Hjelmeland et Chrambach, 1984; Helenius et al., 1979)**.** These detergents can be eliminated by dialysis without affecting the protein activity (Hjelmeland et Chrambach, 1984)**.** The main aim of this study was to find a non-denaturing method to solubilize the main autolysin (PA49.5) of *L. lactis* subsp. *cremoris* ATCC 9596 (Mc5) so as to ensure its purification from the supernatant of cellular remnants and whole cells.

In several studies it was found that autolysins are linked to lipids or other membranous components during the extraction process and especially during the purification. They are therefore considered to be hydrophobic enzymes; however, it is often unclear whether or not the enzyme of interest is in fact hydrophobic. Although several classic and more modern methods are available to solve these kinds of challenges, there is still much uncertainty, and further studies are still needed to clearly resolve the purification of cell membrane hydrophobic enzymes.

An important observation, reported by Brown et al (1970), is that autolysins are associated to teichoic acid in the cell wall of *Bacillus subtilis*. These authors propose that this acid can be eliminated by ethanol precipitation and by gel filtration on an agarose column. Their finding did not confirm this hypothesis, in fact, they found that only 5% of the techoic acid was eliminated using this method. This acid also persisted after multiple washes with ethanol or ether, and it persisted after separation in a polyacrylamide gradient density gel electrophoresis, ion exchange chromatography and gel filtration using 7M urea.

After many attempts, certain autolysins were purified by electrophoresis using SDS as the detergent (*N*-acétylglucosaminidase, ac-*N*-acetylmuramidase and ac-*N*-acetylmuramyl-Lalanine amidase) (Motoyuki et al. 1995). Brown (1972) suggests that the purification of autolysines from lactic bacteria is only possible in the presence of SDS. While SDS is an excellent approach to the purification of this type of enzyme, we propose that another method must be found in order to eliminate the compounds that bind to the autolysin thereby rendering it insoluble. CHAPS, a zwiterrionic detergent, used at high temperatures was evaluated as a potentially more efficient detergent in the purification of this enzyme.

The Problems Associated with Enzyme Purification 25

The autolysins identified in the peak 1 were evaluated in order to determine whether they are acidic or basic proteins. The Davis gel electrophoresis, which allows the identification of acidic proteins, revealed autolytic activity of a band migrating to molecular weight 49.5. The Reisfeld gel that allows the identification of basic proteins revealed no autolytic activity. We can therefore conclude that autolysin P49.5 is an acidic or neutral protein. Since the protein was in the upper area of the Davis gel it is more probable that the

Fig. 1. Gel diffusion assay of HPLC peak 1 on agarose containing cell walls as autolysin

Fig. 2. Autolytic activity of PA49.5 in fraction 1 obtained by HPLC after 48 hours of

method is required if a large quantity of the enzyme in question is to be purified.

We determined that the concentration of protein present in 575 mg of fraction 1 extract diluted in 250 ml of buffer was 0.17 mg/ml. Using the following formula, we calculated that proteins constitute only 7.4% of the fraction 1 extract. This again demonstrates that a better

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ሻ݉݃ሺݐܽܿݎݐݔ݀݁݁ݑݎ݂ܿݐ݊ݑ݉ܣ

protein tends to be more of a neutral pH.

substrate.

renaturation.

ൌ ݁݅݊ݐݎܲΨ

We will present our attempts to purify the enzymes using these various strategies and the problems encountered during certain steps. Some approaches were more problematic that others while some hold more promise in the purification of the enzyme in question.

Before looking at the specific details of how we attempted to purify this autolysin, we will present the assay we used throughout these experiments to verify the presence and purity of our enzyme. For all extractions, a protein profile was examined by SDS-PAGE stained with Coomassie blue. Molecular weight markers were included in all gels. In order to identify autolysins in the preparations, a sample was analyzed by a denaturing SDS-PAGE containing intact cell walls from *Lactococcus lactis* sp *cremoris* MC5 as the substrate for the autolysin according to the method of Potvin et al. (1988). The gel was renatured in buffer containing Triton X100, and enzyme activity was noted by the presence of a clear zone surrounding the enzyme band after staining with Coomassie blue. Proteins were quantified using the method of Lowry. It is also possible to test for general autolysin activity by agarose gel diffusion in a Petri plate. The gel contains cell wall fragments and the fractions containing the autolysin are introduced into wells perforated into the agarose. The gel is incubated and the presence of autolysins is then observed by the presence of a clear zone surrounding the well.

Three different methods of preparing cell extracts were tried in this work, either using autolysis, cell debris or whole cells. We initially started with a crude cell extract prepared by autolysis and the mechanical breakage of the cell. Washed and precipitated cells were suspended in a citrate lysis buffer containing 2% NaCl (w/v) and 0,3M LiCl. The cells were incubated at 30°C for 48 hours to ensure maximum autolysis. The cell debris precipitated by centrifugation was ground with alumina and the supernatant was combined to the first supernatant. These combined supernatants were dialyzed to eliminate the salts, the dialysate was filter sterilized and lyophilized and the extract was then concentrated 28X. This crude extract (CE) was then separated by preparative HPLC which separates the proteins by molecular weight. High molecular weight proteins are found in the first fractions, in the mobile phase. The fractions collected were dialyzed and lyophilized. Autolytic activity of the HPLC fractions was determined by SDS-PAGE with the cell wall substrate.

Four peaks were observed in the HPLC separation profile, the first and largest peak had a retention time of 6 minutes and contained proteins with molecular weights greater than 30 kDa and represented 57,45% of the total estimated area. The autolysin PA49,5 was found in the first fraction as shown in figure 1. C5 represents the results from *Lactococcus lactis* subsp. *cremoris* ATCC 9596 denoted MC5. A visible clear zone can be seen surrounding the wells. Figure 2 presents the results of a denaturing polyacrylamide gel containing cell walls as substrate to identify specific autolysins separated by molecular weight during the electrophoretic process. We can see on figure 2 that there is one band of activity for a protein with the molecular weight of 49.5.

In comparing the protein profile of the crude extract with the proteins in fraction 1, we saw that fraction 1 had approximately 40% less protein than the crude extract indicating that a certain level of purification had been obtained. While the starting crude extract was concentrated 28x, we required 30 µg of fraction 1 proteins to observe autolytic activity. This confirms the limitations of molecular sieving as a method of purifying autolysins.

We will present our attempts to purify the enzymes using these various strategies and the problems encountered during certain steps. Some approaches were more problematic that

Before looking at the specific details of how we attempted to purify this autolysin, we will present the assay we used throughout these experiments to verify the presence and purity of our enzyme. For all extractions, a protein profile was examined by SDS-PAGE stained with Coomassie blue. Molecular weight markers were included in all gels. In order to identify autolysins in the preparations, a sample was analyzed by a denaturing SDS-PAGE containing intact cell walls from *Lactococcus lactis* sp *cremoris* MC5 as the substrate for the autolysin according to the method of Potvin et al. (1988). The gel was renatured in buffer containing Triton X100, and enzyme activity was noted by the presence of a clear zone surrounding the enzyme band after staining with Coomassie blue. Proteins were quantified using the method of Lowry. It is also possible to test for general autolysin activity by agarose gel diffusion in a Petri plate. The gel contains cell wall fragments and the fractions containing the autolysin are introduced into wells perforated into the agarose. The gel is incubated and the presence of autolysins is then observed by the

Three different methods of preparing cell extracts were tried in this work, either using autolysis, cell debris or whole cells. We initially started with a crude cell extract prepared by autolysis and the mechanical breakage of the cell. Washed and precipitated cells were suspended in a citrate lysis buffer containing 2% NaCl (w/v) and 0,3M LiCl. The cells were incubated at 30°C for 48 hours to ensure maximum autolysis. The cell debris precipitated by centrifugation was ground with alumina and the supernatant was combined to the first supernatant. These combined supernatants were dialyzed to eliminate the salts, the dialysate was filter sterilized and lyophilized and the extract was then concentrated 28X. This crude extract (CE) was then separated by preparative HPLC which separates the proteins by molecular weight. High molecular weight proteins are found in the first fractions, in the mobile phase. The fractions collected were dialyzed and lyophilized. Autolytic activity of the HPLC fractions was determined by SDS-PAGE with

Four peaks were observed in the HPLC separation profile, the first and largest peak had a retention time of 6 minutes and contained proteins with molecular weights greater than 30 kDa and represented 57,45% of the total estimated area. The autolysin PA49,5 was found in the first fraction as shown in figure 1. C5 represents the results from *Lactococcus lactis* subsp. *cremoris* ATCC 9596 denoted MC5. A visible clear zone can be seen surrounding the wells. Figure 2 presents the results of a denaturing polyacrylamide gel containing cell walls as substrate to identify specific autolysins separated by molecular weight during the electrophoretic process. We can see on figure 2 that there is one band of activity for a protein

In comparing the protein profile of the crude extract with the proteins in fraction 1, we saw that fraction 1 had approximately 40% less protein than the crude extract indicating that a certain level of purification had been obtained. While the starting crude extract was concentrated 28x, we required 30 µg of fraction 1 proteins to observe autolytic activity. This

confirms the limitations of molecular sieving as a method of purifying autolysins.

others while some hold more promise in the purification of the enzyme in question.

presence of a clear zone surrounding the well.

the cell wall substrate.

with the molecular weight of 49.5.

The autolysins identified in the peak 1 were evaluated in order to determine whether they are acidic or basic proteins. The Davis gel electrophoresis, which allows the identification of acidic proteins, revealed autolytic activity of a band migrating to molecular weight 49.5. The Reisfeld gel that allows the identification of basic proteins revealed no autolytic activity. We can therefore conclude that autolysin P49.5 is an acidic or neutral protein. Since the protein was in the upper area of the Davis gel it is more probable that the protein tends to be more of a neutral pH.

Fig. 1. Gel diffusion assay of HPLC peak 1 on agarose containing cell walls as autolysin substrate.

Fig. 2. Autolytic activity of PA49.5 in fraction 1 obtained by HPLC after 48 hours of renaturation.

We determined that the concentration of protein present in 575 mg of fraction 1 extract diluted in 250 ml of buffer was 0.17 mg/ml. Using the following formula, we calculated that proteins constitute only 7.4% of the fraction 1 extract. This again demonstrates that a better method is required if a large quantity of the enzyme in question is to be purified.

> ͳͲͲݔሿ݉݁ݑ݈ݒ݊݅ݐݑ݈݀݅ݔ݊݅ݐܽݎݐ݊ܿ݁݊݁݅݊ܿݐݎሾ ൌ ݁݅݊ݐݎܲΨ ሻ݉݃ሺݐܽܿݎݐݔ݀݁݁ݑݎ݂ܿݐ݊ݑ݉ܣ

The Problems Associated with Enzyme Purification 27

content of fraction 0-20% is also quite low and the concentration of proteins increases with

The whole cell extract was also precipitated using ammonium sulfate to compare the autolysin profile with that obtained from the HPLC peak 1. The results are presented in figure 5 for each of the four collected fractions at two different protein concentrations. Autolysin PA49.5 is the primary autolysin extracted and it is present in all fractions except the 40-60% fraction (lanes 3 and 7). The intensity of the band in fraction 60-80% (lanes 4 and 8) is approximately half that of the fractions 0-20% and 20-40% and 40-60%. Once again the control does not contain the autolysin due to a low concentration of autolysin in the initial extract. This also demonstrates that ammonium sulfate precipitation not only separates the proteins but also concentrates them. The fact that we found the autolysin in many fractions does however indicate the limitation of this method of its purification and the problem encountered by the difficulty in solubilizing this protein. The ideal situation would be to