**Meet the editor**

Sameh Magdeldin is senior post doctoral researcher in medical school, Niigata University, Japan and academic associate professor (Lecturer) in Physiology dept. SCU, Egypt. He received his M.V.Sc and Ph.D in Physiology and expecting his 2nd Ph.D in proteomics by July, 2012. He is expertise in shot-gun proteomics analysis, reversed- phase chromatography and label- free compar-

ative proteomic approaches. Dr. Magdeldin created the outstanding "All and None" methodology for analyzing large throughput proteomic data published in a highly respected proteomics journal. Beside his published articles, he currently serves as a guest editor, associate editor and a peerreviewer in several international journals. Dr. Magdeldin received several grants and awards such as 8th HUPO congress young investigator award, JSN award, Grant- in – aid for young scientist, and young researcher overseas grant from Japan society for the promotion of science JSPS.

Contents

**Preface IX**

**Part 1 Principles and Applications of Affinity Chromatography 1**

Chapter 2 **Affinity Interactions as a Tool for Protein Immobilization 29**

Chapter 4 **The Difference of Lectin Recovery by Sugar-Bound Resin 75**

Takao Yoshida, Tadashi Maruyama and Hisao Kamiya

**for Purification of IgG from Human Plasma 107**

Mitsuru Jimbo, Shin Satoh, Hirofumi Hasegawa, Hiroshi Miyake,

**Use of S- Substituted Glutathiones as Affinity Ligands 127**  Ramavati Pal, Milana Blakemore, Michelle Ding and Alan G. Clark

Chapter 1 **Affinity Chromatography: Principles and Applications 3**

Sameh Magdeldin and Annette Moser

Eva Benešová and Blanka Králová

**Part 2 Lectin Affinity Chromatography 47**

Chapter 3 **Affinity Chromatography of Lectins 49**

**Part 3 Immunoaffinity Purification 89**

Chapter 6 **Affinity Chromatography**

Jure Pohleven, Borut Štrukelj and Janko Kos

Chapter 5 **Immunoaffinity Chromatography: A Review 91**  Daad A. Abi-Ghanem and Luc R. Berghman

> Lucia Hofbauer, Leopold Bruckschwaiger, Harald Arno Butterweck and Wolfgang Teschner

**Part 4 GST – Tagged Affinity Chromatography 125**

**Proteome of** *Drosophila melanogaster***:**

Chapter 7 **A Study of the Glutathione Transferase**

### Contents

### **Preface XIII**

	- **Part 2 Lectin Affinity Chromatography 47**

#### **Part 3 Immunoaffinity Purification 89**

	- **Part 4 GST Tagged Affinity Chromatography 125**

X Contents

Contents VII

**Part 9 Practical Application**

Chapter 16 **Affinity-Based Methods**

Chapter 15 **The Value of Fungal Protease Inhibitors in Affinity Chromatography 307**

**of Affinity Chromatography in Research Field 305**

Jerica Sabotič, Katarina Koruza, Boštjan Gabor, Matjaž Peterka, Miloš Barut, Janko Kos and Jože Brzin

**for the Separation of Parasite Proteins 333**

C.R. Alves, F.S. Silva, F.O. Oliveira Jr, B.A.S. Pereira, F.A. Pires and M.C.S. Pereira

Piotr Draczkowski, Dariusz Matosiuk and Krzysztof Jozwiak

### **Part 9 Practical Application of Affinity Chromatography in Research Field 305**

### Chapter 15 **The Value of Fungal Protease Inhibitors in Affinity Chromatography 307**  Jerica Sabotič, Katarina Koruza, Boštjan Gabor, Matjaž Peterka, Miloš Barut, Janko Kos and Jože Brzin

VI Contents

**Part 5 Nucleic Acids Affinity Purification 143**

**Affinity Chromatography (IMAC) 165**

**and Purification of Bacterial Toxin 167**  Luciano Moura Martins and Tomomasa Yano

**Part 7 Usage of Affinity Chromatography in Purification**

**for Purification and Biochemical Analysis of Fungal Cell Wall-Degrading Enzymes 177**

**in Conjunction with Affinity Chromatography**

**for the Identification and Purification of Hydrolytic Enzymes or Enzyme Inhibitors**

Alexander V. Konarev and Alison Lovegrove

**as a Key Tool to Purify Protein Protease**

Adeliana Silva de Oliveira, Luciana Maria Araújo Rabêlo, Adriana Ferreira Uchôa and Ana Heloneida Araújo Morais

Salb Katharina, Schinner Elisabeth and Schlossmann Jens

Piotr Draczkowski, Dariusz Matosiuk and Krzysztof Jozwiak

**from Insects and Plants 187**

**Inhibitors from Plants 211** Elizeu Antunes dos Santos,

Chapter 13 **Identification of cGMP-Kinase Complexes by Affinity Chromatography 255**

**as a Quantitative Tool 273**

**as a Tool for Quantification**

**and Their Protein Targets 275**

**of Interactions Between Drug Molecules**

**Part 8 Affinity Chromatography**

Chapter 14 **Affinity Chromatography**

**of Proteases, Protease Inhibitors, and Other Enzymes 175**

Chapter 8 **RNA Affinity Chromatography 145**  Nehal Thakor and Martin Holcik

Chapter 9 **Ion Metallic Affinity Chromatography**

Chapter 10 **Polyhistidine Affinity Chromatography**

Takumi Takeda

Chapter 12 **Affinity Chromatography**

Chapter 11 **Novel Detection Methods Used**

**Part 6 Immobilized Metallic Ion**

### Chapter 16 **Affinity-Based Methods for the Separation of Parasite Proteins 333**  C.R. Alves, F.S. Silva, F.O. Oliveira Jr, B.A.S. Pereira, F.A. Pires and M.C.S. Pereira

Preface

a single volume.

graduate students and post graduate researchers.

participation, this book won't come to light.

With the huge number of books and publications that utilizes different aspects of affinity chromatography, it is still hard to find a freely accessed book that gathers a solid and concise understanding of affinity principles together with its applications in a single volume. The vision of this book is not more than an attempt to provide an open source single volume demonstrating the concept of affinity chromatography with some of its applications that meets the current throughput screening demands of scientists and researchers. This book "*Affinity chromatography*" starts with introductory chapter that passes through the basic principles and snapshot applications of this analytical technique, followed by a wide ranged practical research chapters utilizing affinity chromatography, written by leading experts worldwide. It is worthy to say that the scope of the information contained in this book is still limited to be covered in

*Affinity chromatography* is aimed mainly at those interested in different analytical and separation techniques, particularly, biochemists, biologists, pharmacists, advanced

Finally, I am grateful to the all experts who showed positive and specific "*affinity*"to participate and share in this book with their valuable experience. Indeed, without their

**Sameh Magdeldin, Ph.D**

Suez Canal University,

Japan

Egypt

Medical School, Niigata University,

Ass. Prof. (Lecturer), Physiology Dept.

Senior Post Doc Researcher and Proteomics Team Leader

### Preface

With the huge number of books and publications that utilizes different aspects of affinity chromatography, it is still hard to find a freely accessed book that gathers a solid and concise understanding of affinity principles together with its applications in a single volume. The vision of this book is not more than an attempt to provide an open source single volume demonstrating the concept of affinity chromatography with some of its applications that meets the current throughput screening demands of scientists and researchers. This book "*Affinity chromatography*" starts with introductory chapter that passes through the basic principles and snapshot applications of this analytical technique, followed by a wide ranged practical research chapters utilizing affinity chromatography, written by leading experts worldwide. It is worthy to say that the scope of the information contained in this book is still limited to be covered in a single volume.

*Affinity chromatography* is aimed mainly at those interested in different analytical and separation techniques, particularly, biochemists, biologists, pharmacists, advanced graduate students and post graduate researchers.

Finally, I am grateful to the all experts who showed positive and specific "*affinity*"to participate and share in this book with their valuable experience. Indeed, without their participation, this book won't come to light.

> **Sameh Magdeldin, Ph.D**  Senior Post Doc Researcher and Proteomics Team Leader Medical School, Niigata University, Japan Ass. Prof. (Lecturer), Physiology Dept. Suez Canal University, Egypt

**Part 1**

**Principles and Applications**

 **of Affinity Chromatography**

## **Part 1**

**Principles and Applications of Affinity Chromatography**

**1**

*1Japan 2Egypt 3USA*

**Affinity Chromatography:**

 **Principles and Applications**

*1Department of Structural Pathology, Institute of Nephrology,*

*2Department of Physiology, Faculty of Veterinary Medicine,*

 *Graduate School of Medical and Dental Sciences, Niigata University,*

*3Department of Chemistry, University of Nebraska at Kearney, Kearney, NE,*

Since the inception of affinity chromatography 50 years ago (Cuatrecasas et al, 1968), traditional purification techniques based on pH, ionic strength, or temperature have been replaced by this sophisticated approach. It has been stated that over 60% of all purification techniques involve affinity chromatography (Lowe, 1996). The wide applicability of this method is based on the fact that any given biomolecule that one wishes to purify usually has an inherent recognition site through which it can be bound by a natural or artificial molecule. Thus, we can say that affinity chromatography is principally based on the

a. Incubation of a crude sample with the affinity support to allow the target molecule in

c. Elution (dissociation and recovery) of the target molecule from the immobilized ligand by altering the buffer conditions so that the binding interaction no longer occurs. Since the beginning of this technique, the term affinity chromatography has raised many controversies among researchers. Some say it would be more accurate if termed bioaffinity chromatography (O'Carra et al, 1974) or hydrophobic affinity (Shaltiel, 1974). Nonetheless, the term affinity chromatography has been expanded to describe a potential method of separating biomolecule mixtures on the basis of specific biological interactions. Recently, a modern form

In 1910, the German scientist, Emil Starkenstein published an article which described the concept of resolving macromolecule complexes via their interactions with an immobilized

molecular recognition of a target molecule by a molecule bound to a column.

of liquid chromatography referred to as "flash chromatography" was introduced.

b. Washing away non-bound sample components from the support.

Affinity purification involves 3 main steps:

**2. History of affinity chromatography**

the sample to bind to the immobilized ligand.

**1. Introduction**

Sameh Magdeldin1,2 and Annette Moser3

 *Suez Canal University, Ismailia,*

## **Affinity Chromatography: Principles and Applications**

Sameh Magdeldin1,2 and Annette Moser3

*1Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, 2Department of Physiology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 3Department of Chemistry, University of Nebraska at Kearney, Kearney, NE, 1Japan 2Egypt 3USA*

### **1. Introduction**

Since the inception of affinity chromatography 50 years ago (Cuatrecasas et al, 1968), traditional purification techniques based on pH, ionic strength, or temperature have been replaced by this sophisticated approach. It has been stated that over 60% of all purification techniques involve affinity chromatography (Lowe, 1996). The wide applicability of this method is based on the fact that any given biomolecule that one wishes to purify usually has an inherent recognition site through which it can be bound by a natural or artificial molecule. Thus, we can say that affinity chromatography is principally based on the molecular recognition of a target molecule by a molecule bound to a column.

Affinity purification involves 3 main steps:

Since the beginning of this technique, the term affinity chromatography has raised many controversies among researchers. Some say it would be more accurate if termed bioaffinity chromatography (O'Carra et al, 1974) or hydrophobic affinity (Shaltiel, 1974). Nonetheless, the term affinity chromatography has been expanded to describe a potential method of separating biomolecule mixtures on the basis of specific biological interactions. Recently, a modern form of liquid chromatography referred to as "flash chromatography" was introduced.

### **2. History of affinity chromatography**

In 1910, the German scientist, Emil Starkenstein published an article which described the concept of resolving macromolecule complexes via their interactions with an immobilized

Affinity Chromatography: Principles and Applications 5

bound molecules, a desorption step is usually performed either 1) specifically using a competitive ligand or 2) non-specifically by changing the media atmosphere (e.g. changing the ionic strength, pH or polarity) (Zachariou, 2008). As the elution is perfomed, the purified

Antibodies were first purified using affinity chromatography in 1951 when Campbell et al. used affinity chromatography to isolate rabbit anti-bovine serum albumin antibodies (Campbell et al, 1951). For their purification, bovine serum albumin was used as the affinity ligand on a cellulose support. Two years later, this technique was expanded to purify mushroom tyrosinase using an immobilized inhibitor of the enzyme (azophenol) (Lerman, 1953). Since then, affinity chromatography is commonly used to purify biomolecules such as

Affinity chromatography is often chosen to purify biomolecules due to its excellent specificity, ease of operation, yield and throughput. In addition, affinity chromatography has the ability to remove pathogens, which is necessary if the purified biomolecules are to be used in clinical applications. The purity and recovery of target biomolecules is controlled by the specificity and binding constant of the affinity ligand. In general, the association constants of affinity ligands used for biomolecule purification range from 103 – 108 M-1 (Janson, J-C, 1984). A common affinity ligand used in these purifications is an antibody, but other affinity ligands such as biomimetic dye-ligands, DNA, proteins and small peptides have been used as well. Figure 2 shows a wide variety of molecules that can be purified by

protein can be collected in a concentrated form.

**3.1 Biomolecules purified by affinity chromatography**

enzymes, recombinant proteins, antibodies, and other biomolecules.

affinity chromatography based on their polarity and volatility.

Fig. 2. Illustration showing different molecules that can be purified using affinity

chromatography.

substrate. This manuscript discussed the influence of chloride on the enzymatic activity of liver α-amylase and opened the door for the early beginnings of this approach by several researchers (Arsenis & McCormick, 1966; Bautz & Hall, 1962; Campbell et al, 1951; Sander et al, 1966). Later on, the term affinity chromatography introduced in 1968 by Pedro Cuatecasas, Chris Anfinsen and Meir Wilchek in an article that briefly described the technique of enzyme purification via immobilized substrates and inhibitors (Cuatrecasas et al, 1968). Other early articles described the activation of a Sepharose matrix using a cyanogen bromide (CNBr) reaction (Axen et al, 1967) and the use of a spacer arm to alleviate steric hindrance (Cuatrecasas et al, 1968).

Affinity chromatography is still developing. It has played a central role in many "Omics" technologies, such as genomics, proteomics and metabolomics. The breakthrough development of affinity liquid chromatography has enabled researchers to explore fields such as protein–protein interactions, post translational modifications and protein degradation that were not possible to be examined previously. Finally, the coupling of reversed phase affinity chromatography with mass spectrometry has ultimately aided in discovery of protein biomarkers.

### **3. Fundamental principles of affinity chromatography**

Separation of a desired protein using affinity chromatography relies on the reversible interactions between the protein to be purified and the affinity ligand coupled to chromatographic matrix. As stated earlier, most of the proteins have an inherent recognition site that can be used to select the appropriate affinity ligand. The binding between the protein of interest and the chosen ligand must be both specific and reversible.

Fig. 1. Typical affinity chromatography purification

A typical affinity purification is shown in Figure 1 and involves several steps. First, samples are applied under conditions that favor maximum binding with the affinity ligand. After sample application, a washing step is applied to remove unbound sustances, leaving the desired (bound) molecule still attached to the affinity support. To release and elute the

substrate. This manuscript discussed the influence of chloride on the enzymatic activity of liver α-amylase and opened the door for the early beginnings of this approach by several researchers (Arsenis & McCormick, 1966; Bautz & Hall, 1962; Campbell et al, 1951; Sander et al, 1966). Later on, the term affinity chromatography introduced in 1968 by Pedro Cuatecasas, Chris Anfinsen and Meir Wilchek in an article that briefly described the technique of enzyme purification via immobilized substrates and inhibitors (Cuatrecasas et al, 1968). Other early articles described the activation of a Sepharose matrix using a cyanogen bromide (CNBr) reaction (Axen et al, 1967) and the use of a spacer arm to alleviate

Affinity chromatography is still developing. It has played a central role in many "Omics" technologies, such as genomics, proteomics and metabolomics. The breakthrough development of affinity liquid chromatography has enabled researchers to explore fields such as protein–protein interactions, post translational modifications and protein degradation that were not possible to be examined previously. Finally, the coupling of reversed phase affinity chromatography with mass spectrometry has ultimately aided in

Separation of a desired protein using affinity chromatography relies on the reversible interactions between the protein to be purified and the affinity ligand coupled to chromatographic matrix. As stated earlier, most of the proteins have an inherent recognition site that can be used to select the appropriate affinity ligand. The binding between the

A typical affinity purification is shown in Figure 1 and involves several steps. First, samples are applied under conditions that favor maximum binding with the affinity ligand. After sample application, a washing step is applied to remove unbound sustances, leaving the desired (bound) molecule still attached to the affinity support. To release and elute the

protein of interest and the chosen ligand must be both specific and reversible.

steric hindrance (Cuatrecasas et al, 1968).

discovery of protein biomarkers.

**3. Fundamental principles of affinity chromatography**

Fig. 1. Typical affinity chromatography purification

bound molecules, a desorption step is usually performed either 1) specifically using a competitive ligand or 2) non-specifically by changing the media atmosphere (e.g. changing the ionic strength, pH or polarity) (Zachariou, 2008). As the elution is perfomed, the purified protein can be collected in a concentrated form.

### **3.1 Biomolecules purified by affinity chromatography**

Antibodies were first purified using affinity chromatography in 1951 when Campbell et al. used affinity chromatography to isolate rabbit anti-bovine serum albumin antibodies (Campbell et al, 1951). For their purification, bovine serum albumin was used as the affinity ligand on a cellulose support. Two years later, this technique was expanded to purify mushroom tyrosinase using an immobilized inhibitor of the enzyme (azophenol) (Lerman, 1953). Since then, affinity chromatography is commonly used to purify biomolecules such as enzymes, recombinant proteins, antibodies, and other biomolecules.

Affinity chromatography is often chosen to purify biomolecules due to its excellent specificity, ease of operation, yield and throughput. In addition, affinity chromatography has the ability to remove pathogens, which is necessary if the purified biomolecules are to be used in clinical applications. The purity and recovery of target biomolecules is controlled by the specificity and binding constant of the affinity ligand. In general, the association constants of affinity ligands used for biomolecule purification range from 103 – 108 M-1 (Janson, J-C, 1984). A common affinity ligand used in these purifications is an antibody, but other affinity ligands such as biomimetic dye-ligands, DNA, proteins and small peptides have been used as well. Figure 2 shows a wide variety of molecules that can be purified by affinity chromatography based on their polarity and volatility.

Fig. 2. Illustration showing different molecules that can be purified using affinity chromatography.

Affinity Chromatography: Principles and Applications 7

Regardless of the type of support used in the affinity purification, several factors must be considered when choosing a support material. These include chemical inertness, chemical

Chemical inertness of the support material requires that the affinity support bind only the molecule of interest and have little or no nonspecific binding. While the specificity is related to the affinity ligand immobilized onto the support, the properties of the support must be chosen to limit the nonspecific binding of other molecules. Supports which have little or no nonspecific binding mimic the properties of the aqueous mobile phase. Therefore, chemically inert support materials are hydrophilic. In addition, most separations are performed in low ionic strength media. As a result, the number of charges on the support

In addition, a support material must be chemically stable under normal operating conditions. This includes resistance to degradation by all enzymes and microbes, elution buffers, regenerating solvents, and cleaning agents that will be used within the column. These stability considerations must also be expanded to the stability of the affinity ligandmatrix linkage. Agarose-based support materials meet all of these requirements as they can be used between pH 3 and 12, are not attacked by enzymes, and are not affected by most aqueous eluants. However, ligand attachment in agarose support materials is often not as

Mechanical stability is another consideration when choosing a chromatographic support material for affinity chromatography. Support materials must be able to withstand the backpressures encountered during normal separations without compressing. While most commercial packing materials meet this requirement, the build-up of particulate contaminants may restrict column flow and lead to high backpressures. Under these pressures, soft porous gel supports such as agarose beads will compress and increase the pressure even further causing collapse of the support structure. More mechanically stable supports (e.g. silica and heavily cross-linked polymers) are able to withstand these high pressures, but the build-up of particulate contaminants should be avoided if at all possible. Particle size is an additional consideration when choosing a support material. Ideally, small particle sizes are desired to limit mass transfer effects and limit band broadening. In addition, smaller particle sizes tend to offer greater surface area of the support material and allow for a larger number of affinity ligands to be immobilized on the surface of the support. Unfortunately, as particle size is decreased, backpressures are increased. In addition, when using smaller particles, the potential for the build-up of particulate contaminants and column fouling is increased. For this reason, in preparative applications large particles (30 – 100 μm) are often used. An alternative method to avoid the potential build-up of particulates is to use an expanded-bed support material as discussed earlier and seen in

Pore size is another item that must be considered when using affinity chromatography since the biomolecules of interest must be able to not only pass through the column but also be able to fully interact with the affinity ligand. Based on the Renkin equation which allows the estimation of the effective diffusion coefficient (Renkin, 1954), the pore diameter should be at least 5 times the diameter of the biomolecule being purified (Gustavsson & Larsson, 2006). Therefore, a typical protein with a 60 Å diameter would need a support with at least a 300 Å

stability, mechanical stability, pore size, and particle size.

should be minimized to prevent nonspecific ionic interactions.

stable, depending on the type of linkage used.

Figure 3.

### **3.2 Components of affinity medium**

When affinity chromatography is used for the purification and separation of large biomolecules from complex mixtures, the support (matrix), spacer arms, and ligand must be considered.

### **3.2.1 Affinity supports (matrix)**

Traditionally, affinity chromatography support materials have consisted of porous support materials such as agarose, polymethacrylate, polyacrylamide, cellulose, and silica. All of these support materials are commercially available and come in a range of particle and pore sizes. Some supports may be available with common affinity ligands already immobilized (e.g. protein A, Cibacron Blue, heparin). Other types of support materials are being developed including nonporous supports, membranes, flow-through beads (perfusion media), monolithic supports, and expanded-bed adsorbents.

Nonporous support materials consist of nonporous beads with diameters of 1- 3 μm. These supports allow for fast purifications, but suffer from low surface areas when compared to traditional porous supports. Membranes used in affinity chromatography also lack diffusion pores which limits surface area, but like the nonporous beads allow for fast separations. Flow-through beads or perfusion media (originally developed for ion-exchange chromatography) have both small and large pores present. The addition of the large flowthrough pores allows substances to be directly transported to the interior of the particle which means only short distances are required for diffusion. Monolithic supports are based on the same principle as perfusion media – they contain both large flow-through pores and small diffusion pores. Expanded-bed adsorbents were designed to prevent column clogging and utilize a reverse in flow to allow for the expansion of the column bed which allows for particulates to flow freely through the column and prevent column fouling. See Figure 3. More information about expanded-bed chromatography can be found in (Mattiasson, 1999).

Fig. 3. Expanded-bed chromatography. In this type of chromatography, elution is performed in a normal packed-bed, but during the adsorption-wash step, the flow is reversed and the column bed expanded. This allows for particulate contaminates to pass freely through the column and prevent column clogging.

When affinity chromatography is used for the purification and separation of large biomolecules from complex mixtures, the support (matrix), spacer arms, and ligand must be

Traditionally, affinity chromatography support materials have consisted of porous support materials such as agarose, polymethacrylate, polyacrylamide, cellulose, and silica. All of these support materials are commercially available and come in a range of particle and pore sizes. Some supports may be available with common affinity ligands already immobilized (e.g. protein A, Cibacron Blue, heparin). Other types of support materials are being developed including nonporous supports, membranes, flow-through beads (perfusion

Nonporous support materials consist of nonporous beads with diameters of 1- 3 μm. These supports allow for fast purifications, but suffer from low surface areas when compared to traditional porous supports. Membranes used in affinity chromatography also lack diffusion pores which limits surface area, but like the nonporous beads allow for fast separations. Flow-through beads or perfusion media (originally developed for ion-exchange chromatography) have both small and large pores present. The addition of the large flowthrough pores allows substances to be directly transported to the interior of the particle which means only short distances are required for diffusion. Monolithic supports are based on the same principle as perfusion media – they contain both large flow-through pores and small diffusion pores. Expanded-bed adsorbents were designed to prevent column clogging and utilize a reverse in flow to allow for the expansion of the column bed which allows for particulates to flow freely through the column and prevent column fouling. See Figure 3. More information about expanded-bed chromatography can be found in (Mattiasson, 1999).

Fig. 3. Expanded-bed chromatography. In this type of chromatography, elution is performed in a normal packed-bed, but during the adsorption-wash step, the flow is reversed and the column bed expanded. This allows for particulate contaminates to pass freely through the

**3.2 Components of affinity medium**

**3.2.1 Affinity supports (matrix)**

column and prevent column clogging.

media), monolithic supports, and expanded-bed adsorbents.

considered.

Regardless of the type of support used in the affinity purification, several factors must be considered when choosing a support material. These include chemical inertness, chemical stability, mechanical stability, pore size, and particle size.

Chemical inertness of the support material requires that the affinity support bind only the molecule of interest and have little or no nonspecific binding. While the specificity is related to the affinity ligand immobilized onto the support, the properties of the support must be chosen to limit the nonspecific binding of other molecules. Supports which have little or no nonspecific binding mimic the properties of the aqueous mobile phase. Therefore, chemically inert support materials are hydrophilic. In addition, most separations are performed in low ionic strength media. As a result, the number of charges on the support should be minimized to prevent nonspecific ionic interactions.

In addition, a support material must be chemically stable under normal operating conditions. This includes resistance to degradation by all enzymes and microbes, elution buffers, regenerating solvents, and cleaning agents that will be used within the column. These stability considerations must also be expanded to the stability of the affinity ligandmatrix linkage. Agarose-based support materials meet all of these requirements as they can be used between pH 3 and 12, are not attacked by enzymes, and are not affected by most aqueous eluants. However, ligand attachment in agarose support materials is often not as stable, depending on the type of linkage used.

Mechanical stability is another consideration when choosing a chromatographic support material for affinity chromatography. Support materials must be able to withstand the backpressures encountered during normal separations without compressing. While most commercial packing materials meet this requirement, the build-up of particulate contaminants may restrict column flow and lead to high backpressures. Under these pressures, soft porous gel supports such as agarose beads will compress and increase the pressure even further causing collapse of the support structure. More mechanically stable supports (e.g. silica and heavily cross-linked polymers) are able to withstand these high pressures, but the build-up of particulate contaminants should be avoided if at all possible.

Particle size is an additional consideration when choosing a support material. Ideally, small particle sizes are desired to limit mass transfer effects and limit band broadening. In addition, smaller particle sizes tend to offer greater surface area of the support material and allow for a larger number of affinity ligands to be immobilized on the surface of the support. Unfortunately, as particle size is decreased, backpressures are increased. In addition, when using smaller particles, the potential for the build-up of particulate contaminants and column fouling is increased. For this reason, in preparative applications large particles (30 – 100 μm) are often used. An alternative method to avoid the potential build-up of particulates is to use an expanded-bed support material as discussed earlier and seen in Figure 3.

Pore size is another item that must be considered when using affinity chromatography since the biomolecules of interest must be able to not only pass through the column but also be able to fully interact with the affinity ligand. Based on the Renkin equation which allows the estimation of the effective diffusion coefficient (Renkin, 1954), the pore diameter should be at least 5 times the diameter of the biomolecule being purified (Gustavsson & Larsson, 2006). Therefore, a typical protein with a 60 Å diameter would need a support with at least a 300 Å

Affinity Chromatography: Principles and Applications 9

Fig. 5. Typical structure of an antibody. The amino acids in the Fc region generally have the same sequence, whereas the amino acids in the Fab region have variable amino acid sequences which allows for the specificity of the binding interaction against a wide range of antigens.

Another type of affinity ligand which can be used to purify biomolecules from complex mixtures is a dye-ligand. Dye ligand chromatography originated in 1968 when Haeckel et al. were purifying pyruvate kinase using gel filtration chromatography and found that Blue Dextran (a small dye molecule) co-eluted with the protein (Haeckel et al, 1968). After further investigation, it was determined that binding between the dye and enzyme caused this coelution. The dye-enzyme binding was later utilized in the purification of pyruvate kinase

Biomimetic dye-ligand chromatography takes dye-ligand chromatography one step further and utilizes modified dyes which mimic the natural receptor of the target protein. In addition to offering better binding affinities, these modified dyes were initially developed as a result of the concerns over purity, leakage, and toxicity of the original commercial dyes (Lowe et al, 1992). Cibacron Blue 3GA is one of the most common modified triazine dyes that has been used for protein purification. Its structure can be seen in Figure 6. Covalent attachment of the dye can be achieved through nucleophilic displacement of the dye's chlorine atom by hydroxyl groups on the support's surface (Labrou, 2000; Labrou et al, 1995;

Fig. 6. Chemical structure of blue sepharose dye-ligand (Cibacron Blue 3GA) commonly

used for purification of albumin as well as enzymes (NAD+ and NADP+).

using a Blue Dextran column in 1971 (Staal et al, 1971).

Labrou, 2002).

pore size. Often, the optimal pore size takes into account the ability of the affinity ligand to interact with the biomolecule as well as the surface area of the column since increasing pore size leads to a decrease in the surface area which limits the number of affinity ligands which can be immobilized to the support material.

### **3.2.2 Spacer arms**

Due to the fact that binding sites of the target molecule are sometimes deeply located and difficult to access due to steric hindrance, a spacer arm is often incorporated between the matrix and ligand to facilitate efficient binding and create a more effective and better binding environment. See Figure 4.

Fig. 4. Chromatogram showing better ligation and elution when spacer arms are introduced between the ligand and matrix

The length of these spacer arms is critical. Too short or too long arms may lead to failure of binding or even non-specific binding. In general, the spacer arms are used when coupling molecules less than 1000 Da.

### **3.2.3 Ligands used in affinity chromatography**

Antibodies have several advantages including their high specificity and relatively large binding constants. Antibodies or immunoglobulins are a type of glycoprotein produced when a body's immune system responds to a foreign agent or antigen. Due to the variability of the amino acid sequence in the antibody binding sites (Fab regions shown in Figure 5), it has been estimated that antibodies can be produced for millions or even billions of different foreign agents.

Antibodies which are produced by separate cell lines are referred to as polyclonal antibodies. Monoclonal antibodies are produced when a single antibody producing cell is combined with a carcinoma cell to create a hybridoma which can be grown in a cell culture. Monoclonal antibodies are often more desirable than polyclonal antibodies in affinity chromatography due to their lack of variability which allows for the creation of a more uniform affinity support.

pore size. Often, the optimal pore size takes into account the ability of the affinity ligand to interact with the biomolecule as well as the surface area of the column since increasing pore size leads to a decrease in the surface area which limits the number of affinity ligands which

Due to the fact that binding sites of the target molecule are sometimes deeply located and difficult to access due to steric hindrance, a spacer arm is often incorporated between the matrix and ligand to facilitate efficient binding and create a more effective and better

Fig. 4. Chromatogram showing better ligation and elution when spacer arms are introduced

The length of these spacer arms is critical. Too short or too long arms may lead to failure of binding or even non-specific binding. In general, the spacer arms are used when coupling

Antibodies have several advantages including their high specificity and relatively large binding constants. Antibodies or immunoglobulins are a type of glycoprotein produced when a body's immune system responds to a foreign agent or antigen. Due to the variability of the amino acid sequence in the antibody binding sites (Fab regions shown in Figure 5), it has been estimated that antibodies can be produced for millions or even billions of different

Antibodies which are produced by separate cell lines are referred to as polyclonal antibodies. Monoclonal antibodies are produced when a single antibody producing cell is combined with a carcinoma cell to create a hybridoma which can be grown in a cell culture. Monoclonal antibodies are often more desirable than polyclonal antibodies in affinity chromatography due to their lack of variability which allows for the creation of a more

can be immobilized to the support material.

binding environment. See Figure 4.

between the ligand and matrix

molecules less than 1000 Da.

foreign agents.

uniform affinity support.

**3.2.3 Ligands used in affinity chromatography**

**3.2.2 Spacer arms**

Fig. 5. Typical structure of an antibody. The amino acids in the Fc region generally have the same sequence, whereas the amino acids in the Fab region have variable amino acid sequences which allows for the specificity of the binding interaction against a wide range of antigens.

Another type of affinity ligand which can be used to purify biomolecules from complex mixtures is a dye-ligand. Dye ligand chromatography originated in 1968 when Haeckel et al. were purifying pyruvate kinase using gel filtration chromatography and found that Blue Dextran (a small dye molecule) co-eluted with the protein (Haeckel et al, 1968). After further investigation, it was determined that binding between the dye and enzyme caused this coelution. The dye-enzyme binding was later utilized in the purification of pyruvate kinase using a Blue Dextran column in 1971 (Staal et al, 1971).

Biomimetic dye-ligand chromatography takes dye-ligand chromatography one step further and utilizes modified dyes which mimic the natural receptor of the target protein. In addition to offering better binding affinities, these modified dyes were initially developed as a result of the concerns over purity, leakage, and toxicity of the original commercial dyes (Lowe et al, 1992). Cibacron Blue 3GA is one of the most common modified triazine dyes that has been used for protein purification. Its structure can be seen in Figure 6. Covalent attachment of the dye can be achieved through nucleophilic displacement of the dye's chlorine atom by hydroxyl groups on the support's surface (Labrou, 2000; Labrou et al, 1995; Labrou, 2002).

Fig. 6. Chemical structure of blue sepharose dye-ligand (Cibacron Blue 3GA) commonly used for purification of albumin as well as enzymes (NAD+ and NADP+).

Affinity Chromatography: Principles and Applications 11

A similar process can be used to develop RNA affinity ligands. Once a potential aptamer sequence is identified, it can synthesized *in vitro* and used as the affinity ligand on a chromatographic support. An example of aptamers usage as in purification of L-selectin (Romig et al, 1999) and RNA binding proteins (Dangerfield et al, 2006; Windbichler &

Peptide affinity chromatography is another method which can be used for purifying biomolecules. Peptide affinity ligands are typically identified using one of two techniques (Wang et al, 2004); biological combinatorial peptide libraries (e.g phage-displayed libraries) (Cwirla et al, 1990; Devlin et al, 1990; Smith & Scott, 1993) or solid-phase combinatorial libraries (e.g. one-bead-one-peptide libraries) (Lam et al, 1991). Since then, peptide sequences have been isolated for a wide range of targets (Casey et al, 2008) and have been used to purify staphylococcal enterotoxin B (Wang et al, 2004), β-tryptase (Schaschke et al, 2005), and α-cobratoxin (Byeon & Weisblum, 2004). The main advantages of using peptides

Other ligands can be used in affinity chromatography for biomolecule purification. For more

Immobilization of the affinity ligand is also very important when designing an affinity chromatography method for biomolecule purification. When immobilizing an affinity ligand, care must be taken to ensure that the affinity ligand can actively bind the desired target after the immobilization procedure. Activity of the affinity ligand can be affected by multi-site attachment, orientation of the affinity ligand, and steric hindrance. See Figure 8. Multi-site attachment occurs when an affinity ligand is attached through more than one functional group on a single ligand molecule. If these multiple attachment sites cause the affinity ligand to become denatured or distorted, multisite attachment can lead to reduced binding affinity. However, in some instances, the additional attachment sites can result in more stable ligand attachment. In general, it is best to try for site-specific attachment of the affinity ligands to limit the potential for multi-site attachment. For example, when immobilizing antibodies, covalent attachment is often directed toward the carbohydrate moieties within the Fc region of the antibody. Not only does this limit the number of attachment sites, but it can also help direct the binding and, thus, help orientate the antibody so the binding regions (Fab) are exposed. Another way to prevent multi-site attachment is to use a support that has a limited number of reactive sites. By limiting the reactive sites, the potential for multiple attachments from a single affinity ligand is greatly reduced. A general rule of thumb is the larger the affinity ligand to be immobilized, the

Obviously, when performing affinity purifications, it is important to ensure the affinity ligands are immobilized so that the binding regions are exposed and free to interact and bind with the target molecule(s). Ideally, immobilization methods which specifically avoid attaching the affinity ligand via functional groups within the binding site(s) are used. One way to achieve this when immobilizing proteins is to use site-directed mutagenesis to introduce a single cysteine residue at a site known to be far away from the binding site(s) (Huang et al, 1997).

information on all types of affinity ligands see references (Clonis, 2006; Hage, 2006).

Schroeder, 2006).

as affinity ligands are their low cost and stability.

fewer number of reactive sites on the support needed.

**3.2.4 Immobilization of affinity ligands**

Chlorotriazine polysulfonated aromatic molecules (triazine dyes) have been used for the purification of albumin, oxidoreductases, decarboxylases, glycolytic enzymes, nucleases, hydroloases, lyases, synthetases, and transferases (Labrou, 2000; Labrou et al, 1995). The main advantages of using dye-ligands and biomimetic dye-ligands are their low cost and resistance to chemical and biological degradation. The main disadvantage of these synthetic ligands is that the selection process for a particular biomolecule is empirical and requires extensive screening processes during method development. More information on biomimetic dyes can be found in reference (Clonis et al, 2000).

DNA can also be used as an affinity ligand. It can be used to purify DNA-binding proteins, DNA repair proteins, primases, helicases, polymerases, and restriction enzymes. The scope of biomolecules which can be purified using DNA is expanded when aptamers are utilized. Aptamers are single-stranded oligonucleotides which have a high affinity for a target molecule. SELEX (Systematic Evolution of Ligands by Exponential Enrichment) allows for the isolation of these oligonucleotide sequences and allows for a wide range of potential targets including biomolecules which typically have no affinity for DNA or RNA. The SELEX process for DNA is shown in Figure 7.

Fig. 7. Diagram depicting the SELEX process for the selection of aptamers against a target. First a random short stranded ssDNA (or RNA) library (1014 sequences) is exposed to the target compound and allowed to bind. The unbound oligonucleotides are then separated from the ssDNA-target complexes and removed. The remaining complexes are then disrupted leaving a mixture of aptamer candidates and the target compound. The ssDNA aptamer candidates are then amplified using PCR, the strands separated and the cycle repeated. After multiple cycles (typically 5 – 15), the initial DNA library will have been condensed down to a few sequences which tightly bind the target. These candidates can then be cloned, sequenced and used for affinity chromatography.

Chlorotriazine polysulfonated aromatic molecules (triazine dyes) have been used for the purification of albumin, oxidoreductases, decarboxylases, glycolytic enzymes, nucleases, hydroloases, lyases, synthetases, and transferases (Labrou, 2000; Labrou et al, 1995). The main advantages of using dye-ligands and biomimetic dye-ligands are their low cost and resistance to chemical and biological degradation. The main disadvantage of these synthetic ligands is that the selection process for a particular biomolecule is empirical and requires extensive screening processes during method development. More information on

DNA can also be used as an affinity ligand. It can be used to purify DNA-binding proteins, DNA repair proteins, primases, helicases, polymerases, and restriction enzymes. The scope of biomolecules which can be purified using DNA is expanded when aptamers are utilized. Aptamers are single-stranded oligonucleotides which have a high affinity for a target molecule. SELEX (Systematic Evolution of Ligands by Exponential Enrichment) allows for the isolation of these oligonucleotide sequences and allows for a wide range of potential targets including biomolecules which typically have no affinity for DNA or RNA. The

Fig. 7. Diagram depicting the SELEX process for the selection of aptamers against a target. First a random short stranded ssDNA (or RNA) library (1014 sequences) is exposed to the target compound and allowed to bind. The unbound oligonucleotides are then separated from the ssDNA-target complexes and removed. The remaining complexes are then disrupted leaving a mixture of aptamer candidates and the target compound. The ssDNA aptamer candidates are then amplified using PCR, the strands separated and the cycle repeated. After multiple cycles (typically 5 – 15), the initial DNA library will have been condensed down to a few sequences which tightly bind the target. These candidates can

then be cloned, sequenced and used for affinity chromatography.

biomimetic dyes can be found in reference (Clonis et al, 2000).

SELEX process for DNA is shown in Figure 7.

A similar process can be used to develop RNA affinity ligands. Once a potential aptamer sequence is identified, it can synthesized *in vitro* and used as the affinity ligand on a chromatographic support. An example of aptamers usage as in purification of L-selectin (Romig et al, 1999) and RNA binding proteins (Dangerfield et al, 2006; Windbichler & Schroeder, 2006).

Peptide affinity chromatography is another method which can be used for purifying biomolecules. Peptide affinity ligands are typically identified using one of two techniques (Wang et al, 2004); biological combinatorial peptide libraries (e.g phage-displayed libraries) (Cwirla et al, 1990; Devlin et al, 1990; Smith & Scott, 1993) or solid-phase combinatorial libraries (e.g. one-bead-one-peptide libraries) (Lam et al, 1991). Since then, peptide sequences have been isolated for a wide range of targets (Casey et al, 2008) and have been used to purify staphylococcal enterotoxin B (Wang et al, 2004), β-tryptase (Schaschke et al, 2005), and α-cobratoxin (Byeon & Weisblum, 2004). The main advantages of using peptides as affinity ligands are their low cost and stability.

Other ligands can be used in affinity chromatography for biomolecule purification. For more information on all types of affinity ligands see references (Clonis, 2006; Hage, 2006).

### **3.2.4 Immobilization of affinity ligands**

Immobilization of the affinity ligand is also very important when designing an affinity chromatography method for biomolecule purification. When immobilizing an affinity ligand, care must be taken to ensure that the affinity ligand can actively bind the desired target after the immobilization procedure. Activity of the affinity ligand can be affected by multi-site attachment, orientation of the affinity ligand, and steric hindrance. See Figure 8.

Multi-site attachment occurs when an affinity ligand is attached through more than one functional group on a single ligand molecule. If these multiple attachment sites cause the affinity ligand to become denatured or distorted, multisite attachment can lead to reduced binding affinity. However, in some instances, the additional attachment sites can result in more stable ligand attachment. In general, it is best to try for site-specific attachment of the affinity ligands to limit the potential for multi-site attachment. For example, when immobilizing antibodies, covalent attachment is often directed toward the carbohydrate moieties within the Fc region of the antibody. Not only does this limit the number of attachment sites, but it can also help direct the binding and, thus, help orientate the antibody so the binding regions (Fab) are exposed. Another way to prevent multi-site attachment is to use a support that has a limited number of reactive sites. By limiting the reactive sites, the potential for multiple attachments from a single affinity ligand is greatly reduced. A general rule of thumb is the larger the affinity ligand to be immobilized, the fewer number of reactive sites on the support needed.

Obviously, when performing affinity purifications, it is important to ensure the affinity ligands are immobilized so that the binding regions are exposed and free to interact and bind with the target molecule(s). Ideally, immobilization methods which specifically avoid attaching the affinity ligand via functional groups within the binding site(s) are used. One way to achieve this when immobilizing proteins is to use site-directed mutagenesis to introduce a single cysteine residue at a site known to be far away from the binding site(s) (Huang et al, 1997).

Affinity Chromatography: Principles and Applications 13

Adsorption of affinity ligands may also be used to immobilize affinity ligands onto support materials. The adsorption can be either nonspecific or specific. In nonspecific adsorption the affinity ligand simply adsorbs to the surface of the support material and is a result of Coulombic interactions, hydrogen bonding, and/or hydrophobic interactions. Biospecific adsorption is commonly performed by using avidin or streptavidin for the adsorption of biotin containing affinity ligands or protein A or protein G for the adsorption of antibodies. Both of these immobilization methods allow for site-specific attachment of the affinity ligand which minimizes binding site blockages. When biospecific adsorption is used for immobilization, the primary ligand (i.e. avidin, streptavidin, protein A or protein G) must first be immobilized onto the support material. Avidin, streptavidin, protein A and protein G can be immobilized using amine-reactive methods. Avidin is glycosylated and can also be

Entrapment of affinity ligands was demonstrated by Jackson et al. when human serum albumin (HSA) was entrapped using hydrazide-activated supports and oxidized glycogen as a capping agent (Jackson et al, 2010). Their method can be used with other affinity ligands ranging from 5.8 to 150 kDa. This type of immobilization method is generally less harsh than other immobilization methods and does not require the use of recombinant proteins. In addition, no linkage exists between the affinity ligand and the support which eliminates the

Sol-gel entrapment is another method of encapsulation of affinity ligands (Avnir et al, 2006; Jin & Brennan, 2002; Pierre, 2004). The sol-gel entrapment process is as follows: First, the sol is formed from a silica precursor (e.g. alkoxysilane or glycerated silane). Once the sol has been formed, the buffered protein solution is added and the gelation reaction initiated. This is followed by an aging process in which the sol-gel is dried and further crosslinking of the

Affinity chromatography is currently being used for a wide variety of applications ranging from the study of drug-protein binding interactions to the depletion of high abundance

Affinity chromatography can be used to study drug-protein binding interactions. Frontal analysis, zonal elution, and the Hummel-Dreyer method can be used to measure drugprotein binding constants, to quantify kinetic properties of the various interactions, to quantify allosteric interactions, and to identify drug binding sites. More information about the measurement of drug-protein binding constants can be found in two review articles (Hage, 2002; Hage et al, 2011). Information on quantifying kinetic properties of drug-protein interactions can be found in a review by (Schiel & Hage, 2009). A discussion on the quantification of allosteric interactions by affinity chromatography can be seen in an article by (Chen & Hage, 2004). Additional information on the identification of drug-binding sites

When trying to analyze low abundance proteins, it is often necessary to remove high abundance proteins prior to analysis. This removal effectively enriches low abundance proteins and allows more of them to be identified and quantified. Removal of the top 7 or

silica occurs leaving the protein physically trapped within the cross-linked silica gel.

**4. Current techniques involving affinity chromatography**

proteins to enhance the detection/quantification of dilute proteins.

can be found in a review article (Hage & Austin, 2000).

immobilized through its carbohydrate residues.

potential immobilization problems seen in Figure 8.

Once the cysteine residue is introduced, the protein can be immobilized using a cysteine specific coupling reagent such as N-γ-maleimidobutyryl-oxysuccimide ester.

Fig. 8. Potential immobilization problems which can affect affinity ligand activity by a) multi-site attachment, (b) improper orientation, and (c) steric hindrance.

Affinity ligands can be covalently immobilized, adsorbed onto a surface via nonspecific or biospecific interactions, entrapped within a pore, or coordinated with a metal ion as in metal-ion affinity chromatography (IMAC). Each of these methods has advantages and disadvantages and is briefly discussed below.

Covalent immobilization is one of the most common ways of attaching an affinity ligand to a solid support material. There is a wide range of coupling chemistries available when considering covalent immobilization methods. Amine, sulfhydryl, hydroxyl, aldehyde, and carboxyl groups have been used to link affinity ligands onto support materials. More information about these specific reactions can be found in reference (Kim & Hage, 2006). Although covalent attachment methods are more selective than other immobilization methods, they generally require more steps and chemical reagents. While this may lead to a greater initial cost of preparation, the stability of these supports typically is greater and the support does not need to be periodically regenerated with additional affinity ligands as is typically the case when using adsorption techniques. As a result, covalent immobilization may be more economical in the long-term for the immobilization of costly affinity ligands.

Once the cysteine residue is introduced, the protein can be immobilized using a cysteine

Fig. 8. Potential immobilization problems which can affect affinity ligand activity by a)

Affinity ligands can be covalently immobilized, adsorbed onto a surface via nonspecific or biospecific interactions, entrapped within a pore, or coordinated with a metal ion as in metal-ion affinity chromatography (IMAC). Each of these methods has advantages and

Covalent immobilization is one of the most common ways of attaching an affinity ligand to a solid support material. There is a wide range of coupling chemistries available when considering covalent immobilization methods. Amine, sulfhydryl, hydroxyl, aldehyde, and carboxyl groups have been used to link affinity ligands onto support materials. More information about these specific reactions can be found in reference (Kim & Hage, 2006). Although covalent attachment methods are more selective than other immobilization methods, they generally require more steps and chemical reagents. While this may lead to a greater initial cost of preparation, the stability of these supports typically is greater and the support does not need to be periodically regenerated with additional affinity ligands as is typically the case when using adsorption techniques. As a result, covalent immobilization may be more economical in the long-term for the immobilization of costly affinity ligands.

multi-site attachment, (b) improper orientation, and (c) steric hindrance.

disadvantages and is briefly discussed below.

specific coupling reagent such as N-γ-maleimidobutyryl-oxysuccimide ester.

Adsorption of affinity ligands may also be used to immobilize affinity ligands onto support materials. The adsorption can be either nonspecific or specific. In nonspecific adsorption the affinity ligand simply adsorbs to the surface of the support material and is a result of Coulombic interactions, hydrogen bonding, and/or hydrophobic interactions. Biospecific adsorption is commonly performed by using avidin or streptavidin for the adsorption of biotin containing affinity ligands or protein A or protein G for the adsorption of antibodies. Both of these immobilization methods allow for site-specific attachment of the affinity ligand which minimizes binding site blockages. When biospecific adsorption is used for immobilization, the primary ligand (i.e. avidin, streptavidin, protein A or protein G) must first be immobilized onto the support material. Avidin, streptavidin, protein A and protein G can be immobilized using amine-reactive methods. Avidin is glycosylated and can also be immobilized through its carbohydrate residues.

Entrapment of affinity ligands was demonstrated by Jackson et al. when human serum albumin (HSA) was entrapped using hydrazide-activated supports and oxidized glycogen as a capping agent (Jackson et al, 2010). Their method can be used with other affinity ligands ranging from 5.8 to 150 kDa. This type of immobilization method is generally less harsh than other immobilization methods and does not require the use of recombinant proteins. In addition, no linkage exists between the affinity ligand and the support which eliminates the potential immobilization problems seen in Figure 8.

Sol-gel entrapment is another method of encapsulation of affinity ligands (Avnir et al, 2006; Jin & Brennan, 2002; Pierre, 2004). The sol-gel entrapment process is as follows: First, the sol is formed from a silica precursor (e.g. alkoxysilane or glycerated silane). Once the sol has been formed, the buffered protein solution is added and the gelation reaction initiated. This is followed by an aging process in which the sol-gel is dried and further crosslinking of the silica occurs leaving the protein physically trapped within the cross-linked silica gel.

### **4. Current techniques involving affinity chromatography**

Affinity chromatography is currently being used for a wide variety of applications ranging from the study of drug-protein binding interactions to the depletion of high abundance proteins to enhance the detection/quantification of dilute proteins.

Affinity chromatography can be used to study drug-protein binding interactions. Frontal analysis, zonal elution, and the Hummel-Dreyer method can be used to measure drugprotein binding constants, to quantify kinetic properties of the various interactions, to quantify allosteric interactions, and to identify drug binding sites. More information about the measurement of drug-protein binding constants can be found in two review articles (Hage, 2002; Hage et al, 2011). Information on quantifying kinetic properties of drug-protein interactions can be found in a review by (Schiel & Hage, 2009). A discussion on the quantification of allosteric interactions by affinity chromatography can be seen in an article by (Chen & Hage, 2004). Additional information on the identification of drug-binding sites can be found in a review article (Hage & Austin, 2000).

When trying to analyze low abundance proteins, it is often necessary to remove high abundance proteins prior to analysis. This removal effectively enriches low abundance proteins and allows more of them to be identified and quantified. Removal of the top 7 or

Affinity Chromatography: Principles and Applications 15

In equation 3, the *KD* range is between 10–6 – 10–4 M which means there is more binding and less elution. In equation 4, the *KD* is decreased to 10–1 – 10–2 M by the elution conditions

The interactions described in equations 3 and 4 apply under non-selective (noncompetitive) elution conditions. In case of selective elution or competitive elution, the interaction can be

Fig. 10. Competitive elution of the target by adding a competitive free ligand (triangle).

When adding a competing binding substance or a free ligand (C) that binds to the purified compound of interest during elution, the interaction can be represented as shown in

> *C T CT*

[][] [ ] *<sup>D</sup> C T <sup>K</sup> CT*

where [C] and [T] are the concentration of the free competing ligand and target, respectively

(Graves & Wu, 1974) have shown that the eluted target to total target compound ratio can be

Elutedtarget \* Total boundtarget <sup>1</sup> *DComp* \* 0

where, *p* is the ratio between the volume of competitor added and the pore volume of the gel, *KD* is the dissociation constant for coupled ligand, *KDcomp* is the dissociation constant for

The equilibrium constant, *KD*, is calculated according to equation 6

and [CT] is the concentration of the competing ligand-target complex.

(5)

(7)

(6)

0

*D*

0

*p K L pC <sup>K</sup>*

*p pC*

which results in less binding and more elution of the target compound.

represent as shown in Figure 10.

Equation 5.

represented by equation 7

top 14 high-abundance proteins has been shown to result in a 25% increase in identified proteins (Tu et al, 2010). Moreover, affinity chromatography is widely used in many 'omics' studies (e.g. proteomics, metabolomics and genomics) and is currently used in tandem with other methods to develop high-throughput screening methods for potential drugs.

### **5. Biokinetics of affinity chromatography**

The reaction between the ligand (L) and target compound (T) in an affinity atmosphere (either adsorption or desorption) is represented in Figure 9.

Fig. 9. Basic reaction between compound to be purified and ligand.

The standard definition of the term equilibrium dissociation constant [KD] can be expressed in equation 1,

$$K\_D = \frac{\begin{bmatrix} L \end{bmatrix} \* \begin{bmatrix} T \end{bmatrix}}{\begin{bmatrix} LT \end{bmatrix}} \tag{1}$$

where [L] is the free ligand, [T] is the target compound, and [LT] is the ligand-target complex.

According to the postulation of (Graves & Wu, 1974), the bound target-total target ratio can by represented as shown in equation 2,

$$\frac{\text{Bound target}}{\text{Total bound}} \approx \frac{L\_0}{K\_D} + L\_0 \tag{2}$$

where L0 is the concentration of the ligand (usually 10-4 – 10-2 M). To achieve successful binding, the ratio of bound to total target must be near 1. Therefore, KD should be small compared to ligand concentration. KD can be greatly affected by changing in pH, ionic strength, and temperature. Thus changing these parameters can be used to control the binding and elution efficiency of the reaction and can be expressed as seen in equations 3 and 4,

$$L + T \leftrightharpoons LT \tag{3}$$

$$LT \leftrightharpoons L + T\tag{4}$$

top 14 high-abundance proteins has been shown to result in a 25% increase in identified proteins (Tu et al, 2010). Moreover, affinity chromatography is widely used in many 'omics' studies (e.g. proteomics, metabolomics and genomics) and is currently used in tandem with

The reaction between the ligand (L) and target compound (T) in an affinity atmosphere

The standard definition of the term equilibrium dissociation constant [KD] can be expressed

where [L] is the free ligand, [T] is the target compound, and [LT] is the ligand-target

According to the postulation of (Graves & Wu, 1974), the bound target-total target ratio can

where L0 is the concentration of the ligand (usually 10-4 – 10-2 M). To achieve successful binding, the ratio of bound to total target must be near 1. Therefore, KD should be small compared to ligand concentration. KD can be greatly affected by changing in pH, ionic strength, and temperature. Thus changing these parameters can be used to control the binding and elution efficiency of the reaction and can be expressed as seen in equations 3

> *L T LT*

> *LT L T*

0

*<sup>L</sup> <sup>L</sup> K*

0

*D*

*K*

Boundtarget Total bound *<sup>D</sup>*

 \*

*LT* (1)

(2)

(3)

(4)

*L T*

other methods to develop high-throughput screening methods for potential drugs.

**5. Biokinetics of affinity chromatography**

in equation 1,

complex.

and 4,

by represented as shown in equation 2,

(either adsorption or desorption) is represented in Figure 9.

Fig. 9. Basic reaction between compound to be purified and ligand.

In equation 3, the *KD* range is between 10–6 – 10–4 M which means there is more binding and less elution. In equation 4, the *KD* is decreased to 10–1 – 10–2 M by the elution conditions which results in less binding and more elution of the target compound.

The interactions described in equations 3 and 4 apply under non-selective (noncompetitive) elution conditions. In case of selective elution or competitive elution, the interaction can be represent as shown in Figure 10.

Fig. 10. Competitive elution of the target by adding a competitive free ligand (triangle).

When adding a competing binding substance or a free ligand (C) that binds to the purified compound of interest during elution, the interaction can be represented as shown in Equation 5.

$$\text{C} + T \leftrightharpoons \text{C} \text{T} \tag{5}$$

The equilibrium constant, *KD*, is calculated according to equation 6

$$K\_D = \frac{[C] \* [T]}{[CT]} \tag{6}$$

where [C] and [T] are the concentration of the free competing ligand and target, respectively and [CT] is the concentration of the competing ligand-target complex.

(Graves & Wu, 1974) have shown that the eluted target to total target compound ratio can be represented by equation 7

$$\frac{\text{Euted target}}{\text{Total bound target}} \approx \left(\bigvee\_{p+1}^{p} + 1\right)^{\*} \left| \frac{p\mathbb{C}\_{0}}{p\mathbb{C}\_{0} + \frac{K\_{DComp} \text{ } ^\circ L0}{K\_{D}}} \right| \tag{7}$$

where, *p* is the ratio between the volume of competitor added and the pore volume of the gel, *KD* is the dissociation constant for coupled ligand, *KDcomp* is the dissociation constant for

Affinity Chromatography: Principles and Applications 17

amino acids to entire domains or even whole proteins. Tags can act both as a marker for

The properties of fusion tags allow tagged proteins to be easily manipulated in the laboratory. Most significantly, the well-characterized tag-ligand chemistry enables singlestep affinity purification of tagged molecules using immobilized versions of their corresponding affinity ligands. In addition, antibodies to fusion tags are also available and can be used for "universal" purification and detection of tagged proteins (i.e., without

In general, the most commonly used tags are glutathione-S-transferase (GST), histidine fusion (His or polyHis tag) and protein A fusion tags. Other types of fusion tags are also available including maltose-binding protein (di Guan et al, 1988), thioredoxin (LaVallie et al, 1993), NusA (Whetstone et al, 2004), GB1 domain for protein G (Davis et al, 1999), and others (Balbas, 2001; Thorn et al, 2000). The decision to use any of these tagging methods depends mainly on the needs of of the researcher. Table 1 compares GST and (His)6 tags and

protein expression and to help facilitate protein purification.

Site-specific proteases enable cleavage of tag if required

GST tag easily detected using an

Simple purification. Very mild elution conditions minimize risk of damage to functionality and antigenicity of target proteins

GST tag can help stabilize folding of

utilizing affinity chromatography will be discussed.

recombinant proteins

al, 1999).

having to obtain or develop a probe for each specific recombinant protein).

may help when deciding which tag is appropriate for a particular purification.

**GST tag (His)6 tag** Can be used in any expression system Purification procedure gives high yields of pure product Selection of purification products available for any scale

acids are preferable

conditions if required.

may be necessary

enzyme assay or an immunoassay (His)6 tag easily detected using an immunoassay

Fusion proteins form dimers Small tag is less likely to interfere with structure

Table 1. Comparison of GST and His tags for protein purification. The information from this table is summarized fromthe Amersham recombinant protein handbook and (Geoghegan et

In the following sections, the most commonly used purification techniques and methods

Site-specific proteases enable cleavage of tag if required. N.B. enterokinase sites that enable tag cleavage without leaving behind extra amino

Simple purification, but elution conditions are not as mild as for GST fusion proteins.

Purification can be performed under denaturing

(His)6 -dihydrofolate reductase tag stabilizes

Mass determination by mass spectrometry not always accurate for some (His)6 fusion proteins

Neutral pH but imidazole may cause precipitation. Desalting to remove imidazole

small peptides during expression

and function of fusion partner

the free competing ligand, *C0* is the concentration of the competing ligand (usually 10-2 – 10-1 M) and L0 is the concentration of the coupled ligand, usually 10-4 – 10-2 M.

If both KD and KDcomp are similar, then the concentrations of the competing and coupled ligand must be similar to achieve an efficient elution. On the other hand, if KDcomp is equal to 5\*KD we would expect that the concentration of the competing ligand will need to be 5x higher to achieve successful elution.

### **6. Applications and uses of affinity chromatography**

### **6.1 Immunoglobulin purification (antibody immobilization)**

Antibodies can be immobilized by both covalent and adsorption methods. Random covalent immobilization methods generally link antibodies to the solid support via their free amine groups using cyanogen bromide, N-hydroxysuccinimide, N,N'-carbonyldiimidazole, tresyl chloride, or tosyl chloride. Alternatively, free amine groups can react with aldehyde or free epoxy groups on an activated support. As these are random immobilization methods, the antibody binding sites may be blocked due to improper orientation, multi-site attachment or steric hindrance as shown in Figure 8.

Site-specific covalent immobilization of antibodies can be achieved by converting the carbohydrate residues located in the Fc region of the antibody to produce aldehyde residues which can react with amine or hydrazide supports (Ruhn et al, 1994). Another site-specific immobilization of antibodies can be accomplished by utilizing the free sulfhydryl groups of Fab fragments. These groups can be used to couple the antibody fragments to an affinity support using a variety of established methods including epoxy, divinylsulfone, iodoacetyl, bromoacetyl, thiol, maleimide, TNB-thiol, tresyl chloride, or tosyl chloride methods (Hermanson et al, 1992).

Antibodies can also be immobilized by adsorbing them onto secondary ligands. For example, if an antibody is reacted with hydrazide biotin, the hydrazide can react with oxidized carbohydrate residues on the Fc region of the antibody. The resultant biotinylated antibody can then be adsorbed onto an avidin or streptavidin affinity support. This type of biotin immobilization allows for site-specific immobilization of the antibody and can be performed using commercially available biotinylation kits.

Alternatively, antibodies can be directly adsorbed onto a protein A or protein G support due to the specific interaction of antibodies with protein A and G. Immobilized antibodies on the protein A or G support can easily be replaced by using a strong eluent, regenerating the protein A/G, and re-applying fresh antibodies. Generally, this method is used when a high capacity/high activity support is needed. If a more permanent immobilization is desired, the adsorbed antibodies may be cross-linked to the support material using carbodiimide (Phillips et al, 1985) or dimethyl pimelimidate (Schneider et al, 1982; Sisson & Castor, 1990).

### **6.2 Recombinant tagged proteins**

Purification of proteins can be easier and simpler if the protein of interest is tagged with a known sequence commonly referred to as a tag. This tag can range from a short sequence of

the free competing ligand, *C0* is the concentration of the competing ligand (usually 10-2 – 10-1

If both KD and KDcomp are similar, then the concentrations of the competing and coupled ligand must be similar to achieve an efficient elution. On the other hand, if KDcomp is equal to 5\*KD we would expect that the concentration of the competing ligand will need to be 5x

Antibodies can be immobilized by both covalent and adsorption methods. Random covalent immobilization methods generally link antibodies to the solid support via their free amine groups using cyanogen bromide, N-hydroxysuccinimide, N,N'-carbonyldiimidazole, tresyl chloride, or tosyl chloride. Alternatively, free amine groups can react with aldehyde or free epoxy groups on an activated support. As these are random immobilization methods, the antibody binding sites may be blocked due to improper orientation, multi-site attachment or

Site-specific covalent immobilization of antibodies can be achieved by converting the carbohydrate residues located in the Fc region of the antibody to produce aldehyde residues which can react with amine or hydrazide supports (Ruhn et al, 1994). Another site-specific immobilization of antibodies can be accomplished by utilizing the free sulfhydryl groups of Fab fragments. These groups can be used to couple the antibody fragments to an affinity support using a variety of established methods including epoxy, divinylsulfone, iodoacetyl, bromoacetyl, thiol, maleimide, TNB-thiol, tresyl chloride, or tosyl chloride methods

Antibodies can also be immobilized by adsorbing them onto secondary ligands. For example, if an antibody is reacted with hydrazide biotin, the hydrazide can react with oxidized carbohydrate residues on the Fc region of the antibody. The resultant biotinylated antibody can then be adsorbed onto an avidin or streptavidin affinity support. This type of biotin immobilization allows for site-specific immobilization of the antibody and can be

Alternatively, antibodies can be directly adsorbed onto a protein A or protein G support due to the specific interaction of antibodies with protein A and G. Immobilized antibodies on the protein A or G support can easily be replaced by using a strong eluent, regenerating the protein A/G, and re-applying fresh antibodies. Generally, this method is used when a high capacity/high activity support is needed. If a more permanent immobilization is desired, the adsorbed antibodies may be cross-linked to the support material using carbodiimide (Phillips et al, 1985) or dimethyl pimelimidate (Schneider et al, 1982; Sisson & Castor, 1990).

Purification of proteins can be easier and simpler if the protein of interest is tagged with a known sequence commonly referred to as a tag. This tag can range from a short sequence of

M) and L0 is the concentration of the coupled ligand, usually 10-4 – 10-2 M.

**6. Applications and uses of affinity chromatography 6.1 Immunoglobulin purification (antibody immobilization)**

performed using commercially available biotinylation kits.

higher to achieve successful elution.

steric hindrance as shown in Figure 8.

(Hermanson et al, 1992).

**6.2 Recombinant tagged proteins**

amino acids to entire domains or even whole proteins. Tags can act both as a marker for protein expression and to help facilitate protein purification.

The properties of fusion tags allow tagged proteins to be easily manipulated in the laboratory. Most significantly, the well-characterized tag-ligand chemistry enables singlestep affinity purification of tagged molecules using immobilized versions of their corresponding affinity ligands. In addition, antibodies to fusion tags are also available and can be used for "universal" purification and detection of tagged proteins (i.e., without having to obtain or develop a probe for each specific recombinant protein).

In general, the most commonly used tags are glutathione-S-transferase (GST), histidine fusion (His or polyHis tag) and protein A fusion tags. Other types of fusion tags are also available including maltose-binding protein (di Guan et al, 1988), thioredoxin (LaVallie et al, 1993), NusA (Whetstone et al, 2004), GB1 domain for protein G (Davis et al, 1999), and others (Balbas, 2001; Thorn et al, 2000). The decision to use any of these tagging methods depends mainly on the needs of of the researcher. Table 1 compares GST and (His)6 tags and may help when deciding which tag is appropriate for a particular purification.

Table 1. Comparison of GST and His tags for protein purification. The information from this table is summarized fromthe Amersham recombinant protein handbook and (Geoghegan et al, 1999).

In the following sections, the most commonly used purification techniques and methods utilizing affinity chromatography will be discussed.

Affinity Chromatography: Principles and Applications 19

Histidine tags have strong affinity for metal ions (e.g. Co2+, Ni2+, Cu2+, and Zn2+). One of the first support materials used immobilized iminodiacetic acid which can bind metal ions and allow for the coordination complex with the His-tagged protein. One difficulty with iminodiacetic acid supports is the potential for metal ion leaching leading to a decreased protein yield. Modern support materials, including nickel-nitrilotriacetic acid (Ni-NTA) and cobalt-carboxymethylasparate (Co-CMA), show limited leaching and, therefore, result in more efficient protein purifications. The coordination of a His-tag with a Ni-NTA support can be seen in Figure 12. Once the tagged protein is bound by the immobilized chelating agent, it can be eluted by introducing a competing agent for the chelating group (imidazole)

Fig. 12. showing the complex formed between the poly-histidine tag and a nickel NTA

bodies and must be denatured so they can be solubilized.

One advantage of using His-tags for protein purification includes the small size of the affinity ligand. Due to the small size, it has minimal effects on the folding of the protein. In addition, if the His-tag is placed on the N-terminal end of the protein, it can easily be removed using an endoprotease. Another advantage of using His-tag purification methods is that polyhistidine tags can bind proteins under both native and denaturing conditions. The use of denaturing conditions becomes important when proteins are found in inclusion

Disadvantages of using His-tag protein purification include potential degradation of the Histag, dimer and tetramer formation, and coelution of other histidine-containing proteins. First, when a few histidine residues are proteolytically degraded, the affinity of the tagged protein is greatly reduced leading to a decrease in the protein yield. Second, once a protein has a His-tag added to its structure, it has the potential to form dimers and tetramers in the presence of metal ions. While this is often not a large problem, it can lead to inaccurate molecular mass estimates of the tagged protein. A third disadvantage of protein purification using His-tags is

coelution of proteins that naturally have two or more adjacent histidine residues.

or an additional metal chelating agent (EDTA).

support.

### **6.2.1 GST tagged purification**

Glutathione S-transferase (GST) is a 26 kDa protein (211 amino acids) located in cytosole or mitochondria and present both in eukaryotes and prokaryotes (e.g. *Schistosoma japonicum*). The enzymes have various sources both native and recombinantly expressed by fusion to the N-terminus of target proteins (Allocati et al, 2009; Allocati et al, 2011; Sheehan et al, 2001; Udomsinprasert et al, 2005). GST-fusion proteins can also be produced in *Escherichia coli* as recombinant proteins. Separation and purifcation of GST-tagged proteins is possible since the GST tag is capable of binding its substrate, glutathione (tripeptide, Glu-Cys-Gly).

When glutathione is reduced (GSH), it can be immobilized onto a solid support through its sulfhydryl group. This property can be used to crosslink glutathione with agarose beads and, thus, can be used to capture pure GST or GST-tagged proteins via the enzyme-substrate binding reaction (Beckett & Hayes, 1993; Douglas, 1987). Binding is most efficient near neutral physiological conditions (pH 7.5) using Tris saline buffer and mild conditions to preserve the structure and enzymatic function of GST. As a result of the potential for permanent denaturation, denaturing elution conditions are not compatible with GST purification. In addition, upon denaturation or reduction, the structure of the GST fusion tag often degrades.

Following a washing step to remove unbound samples, the bound GST-fusion protein can be recovered by the addition of excess reduced glutathione since the affinity of GST for free glutathione is higher than the affinity for immobilized glutathione. The free glutathione replaces the immobilized glutathione and releases the GST-tagged protein from the matrix allowing its elution from the column.

Fig. 11. GST- tagged protein immobilization.

### **6.2.2 His-tagged protein purification**

Recombinant proteins which have histidine tags can be purified using immobilized metal ion chromatography (IMAC). The His-tag can be placed on either the N- or C-terminus. Optimal binding and, therefore, purification efficiency is achieved when the His-tag is freely accessible to metal ion support (Dong et al, 2010).

Glutathione S-transferase (GST) is a 26 kDa protein (211 amino acids) located in cytosole or mitochondria and present both in eukaryotes and prokaryotes (e.g. *Schistosoma japonicum*). The enzymes have various sources both native and recombinantly expressed by fusion to the N-terminus of target proteins (Allocati et al, 2009; Allocati et al, 2011; Sheehan et al, 2001; Udomsinprasert et al, 2005). GST-fusion proteins can also be produced in *Escherichia coli* as recombinant proteins. Separation and purifcation of GST-tagged proteins is possible since

When glutathione is reduced (GSH), it can be immobilized onto a solid support through its sulfhydryl group. This property can be used to crosslink glutathione with agarose beads and, thus, can be used to capture pure GST or GST-tagged proteins via the enzyme-substrate binding reaction (Beckett & Hayes, 1993; Douglas, 1987). Binding is most efficient near neutral physiological conditions (pH 7.5) using Tris saline buffer and mild conditions to preserve the structure and enzymatic function of GST. As a result of the potential for permanent denaturation, denaturing elution conditions are not compatible with GST purification. In addition, upon denaturation or reduction, the structure of the GST fusion tag

Following a washing step to remove unbound samples, the bound GST-fusion protein can be recovered by the addition of excess reduced glutathione since the affinity of GST for free glutathione is higher than the affinity for immobilized glutathione. The free glutathione replaces the immobilized glutathione and releases the GST-tagged protein from the matrix

Recombinant proteins which have histidine tags can be purified using immobilized metal ion chromatography (IMAC). The His-tag can be placed on either the N- or C-terminus. Optimal binding and, therefore, purification efficiency is achieved when the His-tag is freely

the GST tag is capable of binding its substrate, glutathione (tripeptide, Glu-Cys-Gly).

**6.2.1 GST tagged purification**

often degrades.

allowing its elution from the column.

Fig. 11. GST- tagged protein immobilization.

accessible to metal ion support (Dong et al, 2010).

**6.2.2 His-tagged protein purification**

Histidine tags have strong affinity for metal ions (e.g. Co2+, Ni2+, Cu2+, and Zn2+). One of the first support materials used immobilized iminodiacetic acid which can bind metal ions and allow for the coordination complex with the His-tagged protein. One difficulty with iminodiacetic acid supports is the potential for metal ion leaching leading to a decreased protein yield. Modern support materials, including nickel-nitrilotriacetic acid (Ni-NTA) and cobalt-carboxymethylasparate (Co-CMA), show limited leaching and, therefore, result in more efficient protein purifications. The coordination of a His-tag with a Ni-NTA support can be seen in Figure 12. Once the tagged protein is bound by the immobilized chelating agent, it can be eluted by introducing a competing agent for the chelating group (imidazole) or an additional metal chelating agent (EDTA).

Fig. 12. showing the complex formed between the poly-histidine tag and a nickel NTA support.

One advantage of using His-tags for protein purification includes the small size of the affinity ligand. Due to the small size, it has minimal effects on the folding of the protein. In addition, if the His-tag is placed on the N-terminal end of the protein, it can easily be removed using an endoprotease. Another advantage of using His-tag purification methods is that polyhistidine tags can bind proteins under both native and denaturing conditions. The use of denaturing conditions becomes important when proteins are found in inclusion bodies and must be denatured so they can be solubilized.

Disadvantages of using His-tag protein purification include potential degradation of the Histag, dimer and tetramer formation, and coelution of other histidine-containing proteins. First, when a few histidine residues are proteolytically degraded, the affinity of the tagged protein is greatly reduced leading to a decrease in the protein yield. Second, once a protein has a His-tag added to its structure, it has the potential to form dimers and tetramers in the presence of metal ions. While this is often not a large problem, it can lead to inaccurate molecular mass estimates of the tagged protein. A third disadvantage of protein purification using His-tags is coelution of proteins that naturally have two or more adjacent histidine residues.

Affinity Chromatography: Principles and Applications 21

biotinylation sequence into a recombinant protein. Biotin protein ligase can then be used to add biotin in a post-translational modification step (Cronan & Reed, 2000). Biotin, also known as vitamin H or vitamin B7, is a relatively small cofactor present in cells. In affinity chromatography it is often used an affinity tag due to its very strong interactions with avidin and streptavidin. One advantage of using biotin as an affinity tag is that it has a

Streptavidin is a large protein (60 kDa) that can be obtained from *Streptomyces avidinii* and bind biotin with an affinity constant of 1013 M–1. Avidin is a slightly larger glycoprotein (66 kDa) with slightly stronger binding to biotin (1015 M–1). Both avidin and streptavidin have four subunits that can each bind one biotin molecule. To purify biotinylated biomolecules, streptavidin is immobilized onto a support material and used to extract the biotinylated molecules out of solution. Both avidin and streptavidin may be immobilized using aminereactive coupling chemistries. In addition, avidin can also be immobilized via its

Due to the strong interaction between biotin and (strept)avidin, harsh elution conditions are required to disrupt the binding. For example, 6 M guanidine hydrochloride at pH 1.5 is commonly used to elute the bound biotinylated biomolecule. This prevents the recovery of most proteins in their active form. To overcome this difficulty, modified (strept)avidin or modified biotin may be used to create a lower affinity interaction. In one study chemically modified avidin had relatively strong binding (>109 M–1), but was also able to completely release biotinylated molecules at pH 10 (Morag et al, 1996). In addition, at any pH between 4 and 10, a 0.6 mM biotin solution could be used to displace and elute the biotinylated

Biotin is also used in isotopically coded affinity tags (ICATs) which can be used to compare the protein content in two different samples (Bottari et al, 2004). The ICAT consists of two labels, one which contains deuterium (heavy) and one which contains only hydrogen (light). The two labels (light and heavy) are added separately to the cell lysates being compared. Since the reagent contains a thiol-specific reactive group, it will covalently bind free cysteines on proteins. The labeled lysates are combined, digested with trypsin, and then isolated on a streptavidin column. After a second separation step, the labeled proteins are analyzed using mass spectrometry. The change in protein expression between the two cell lysates can then be quantified and related to the different conditions applied to the two sets

Affinity purification is a helpful tool for cleaning up and removing excess albumin and α2 macroglobulin contamination from samples since these components can mask or interfere with subsequent steps of analysis (e.g. mass spectrometry and immunoprecipitation). One purification method which can be used to remove these contaminants either before or after other purification steps is Blue sepharose affinity chromatography. In this method, the dye ligand is covalently coupled to sepharose via a chlorotriazine ring. Albumin binds in a nonspecific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand (Antoni et al, 1978; Peters et al, 1973; Travis & Pannell, 1973; Young & Webb, 1978). The most commonly used dye is Cibacron blue F-3-GA which can be immobilized onto

**6.5 Affinity purification of albumin and macroglobulin contamination**

minimal effect on the activity of a large biomolecule due to its small size (244 Da).

carbohydrate residues.

molecules.

of cell lysates.

### **6.3 Protein A, G, and L purification**

Proteins A, G, and L are native or recombinant proteins of microbial origin which bind specifically to immunoglobulins including immunoglobulin G (IgG). IgG represents 80% of serum immunoglobulins. Native and recombinant protein A can be cloned in *Staphylococcus aureus*. Recombinant protein G (cell surface protein) is cloned in *Streptococcus* while recombinant protein L is cloned from *Peptostreptococcus magnus*. Both protein A and G specifically bind the Fc region of IgG while protein L binds to the kappa light chains of IgG.

The most popular matrixes or supports for affinity applications which utilize protein A, G, or L is beaded agarose (e.g. Sepharose CL-4B; agarose crosslinked with 2,3 dibromopropanol and desulphated by alkaline hydrolysis under reductive conditions), polyacrylamide, and magnetic beads (Grodzki & Berenstein, 2010; Hober et al, 2007; Katoh et al, 2007; Tyutyulkova & Paul, 1995).

All three proteins bind extensively with the IgG subclass. In general, protein A is more suitable for cat, dog, rabbit and pig IgG whereas protein G is generally more preferable when purifying mouse or human IgG. A combination of protein A and G is also applicable for purifying a wide range of mammalian IgG samples. Since protein L binds to the kappa light chain of immunoglobulins and these light chains exist in other immunoglobulins (i.e IgG, IgM, IgA, and IgE), protein L is suitable for the purification of different classes of antibodies. The binding characteristics of antibody binding proteins (A, G and L) to a variety immunoglobulin species is summarized in Table 2. IgGs from most species bind to protein A and G near physiological pH and ionic strength. To elute purified immunoglobulins from protein G sepharose, the pH should be less than 2.7.

\*Binding affinity based on total IgG binding, L proteins binds to Kappa light chains while Proteins A and G bind to Fc region.

Table 2. Binding affinity for proteins A, G, and L with a variety of immunoglobulin species.

### **6.4 Biotin and biotinylated molecules purification**

If a biotin tag can be incorporated into a biomolecule, it can be used to purify the biomolecule using a streptavidin or avidin affinity support. One way is to insert a

Proteins A, G, and L are native or recombinant proteins of microbial origin which bind specifically to immunoglobulins including immunoglobulin G (IgG). IgG represents 80% of serum immunoglobulins. Native and recombinant protein A can be cloned in *Staphylococcus aureus*. Recombinant protein G (cell surface protein) is cloned in *Streptococcus* while recombinant protein L is cloned from *Peptostreptococcus magnus*. Both protein A and G specifically bind the Fc region of IgG while protein L binds to the kappa light chains of IgG. The most popular matrixes or supports for affinity applications which utilize protein A, G, or L is beaded agarose (e.g. Sepharose CL-4B; agarose crosslinked with 2,3 dibromopropanol and desulphated by alkaline hydrolysis under reductive conditions), polyacrylamide, and magnetic beads (Grodzki & Berenstein, 2010; Hober et al, 2007; Katoh

All three proteins bind extensively with the IgG subclass. In general, protein A is more suitable for cat, dog, rabbit and pig IgG whereas protein G is generally more preferable when purifying mouse or human IgG. A combination of protein A and G is also applicable for purifying a wide range of mammalian IgG samples. Since protein L binds to the kappa light chain of immunoglobulins and these light chains exist in other immunoglobulins (i.e IgG, IgM, IgA, and IgE), protein L is suitable for the purification of different classes of antibodies. The binding characteristics of antibody binding proteins (A, G and L) to a variety immunoglobulin species is summarized in Table 2. IgGs from most species bind to protein A and G near physiological pH and ionic strength. To elute purified

immunoglobulins from protein G sepharose, the pH should be less than 2.7.

**Species Protein A Protein G Protein L\* Human** Strong Strong Strong **Mouse** Strong Strong Strong **Rat** Weak Medium Strong **Cow** Weak Medium Strong **Goat** Weak Strong No binding **Sheep** Weak Strong No binding **Horse** Weak Strong Unknown **Rabbit** Strong Strong Weak **Guinea pig** Strong Weak Unknown **Pig** Strong Weak Strong **Dog** Strong Weak Unknown **Cat** Strong Weak Unknown **Chicken** Unknown Unknown Unknown

\*Binding affinity based on total IgG binding, L proteins binds to Kappa light chains while Proteins A

Table 2. Binding affinity for proteins A, G, and L with a variety of immunoglobulin species.

If a biotin tag can be incorporated into a biomolecule, it can be used to purify the biomolecule using a streptavidin or avidin affinity support. One way is to insert a

**6.3 Protein A, G, and L purification**

et al, 2007; Tyutyulkova & Paul, 1995).

and G bind to Fc region.

**6.4 Biotin and biotinylated molecules purification**

biotinylation sequence into a recombinant protein. Biotin protein ligase can then be used to add biotin in a post-translational modification step (Cronan & Reed, 2000). Biotin, also known as vitamin H or vitamin B7, is a relatively small cofactor present in cells. In affinity chromatography it is often used an affinity tag due to its very strong interactions with avidin and streptavidin. One advantage of using biotin as an affinity tag is that it has a minimal effect on the activity of a large biomolecule due to its small size (244 Da).

Streptavidin is a large protein (60 kDa) that can be obtained from *Streptomyces avidinii* and bind biotin with an affinity constant of 1013 M–1. Avidin is a slightly larger glycoprotein (66 kDa) with slightly stronger binding to biotin (1015 M–1). Both avidin and streptavidin have four subunits that can each bind one biotin molecule. To purify biotinylated biomolecules, streptavidin is immobilized onto a support material and used to extract the biotinylated molecules out of solution. Both avidin and streptavidin may be immobilized using aminereactive coupling chemistries. In addition, avidin can also be immobilized via its carbohydrate residues.

Due to the strong interaction between biotin and (strept)avidin, harsh elution conditions are required to disrupt the binding. For example, 6 M guanidine hydrochloride at pH 1.5 is commonly used to elute the bound biotinylated biomolecule. This prevents the recovery of most proteins in their active form. To overcome this difficulty, modified (strept)avidin or modified biotin may be used to create a lower affinity interaction. In one study chemically modified avidin had relatively strong binding (>109 M–1), but was also able to completely release biotinylated molecules at pH 10 (Morag et al, 1996). In addition, at any pH between 4 and 10, a 0.6 mM biotin solution could be used to displace and elute the biotinylated molecules.

Biotin is also used in isotopically coded affinity tags (ICATs) which can be used to compare the protein content in two different samples (Bottari et al, 2004). The ICAT consists of two labels, one which contains deuterium (heavy) and one which contains only hydrogen (light). The two labels (light and heavy) are added separately to the cell lysates being compared. Since the reagent contains a thiol-specific reactive group, it will covalently bind free cysteines on proteins. The labeled lysates are combined, digested with trypsin, and then isolated on a streptavidin column. After a second separation step, the labeled proteins are analyzed using mass spectrometry. The change in protein expression between the two cell lysates can then be quantified and related to the different conditions applied to the two sets of cell lysates.

### **6.5 Affinity purification of albumin and macroglobulin contamination**

Affinity purification is a helpful tool for cleaning up and removing excess albumin and α2 macroglobulin contamination from samples since these components can mask or interfere with subsequent steps of analysis (e.g. mass spectrometry and immunoprecipitation). One purification method which can be used to remove these contaminants either before or after other purification steps is Blue sepharose affinity chromatography. In this method, the dye ligand is covalently coupled to sepharose via a chlorotriazine ring. Albumin binds in a nonspecific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand (Antoni et al, 1978; Peters et al, 1973; Travis & Pannell, 1973; Young & Webb, 1978). The most commonly used dye is Cibacron blue F-3-GA which can be immobilized onto

Affinity Chromatography: Principles and Applications 23

Lectin affinity columns can be prepared by immobilizing lectins with different specificities toward oligosaccharides to a variety of matrices, including agarose (West & Goldring, 2004), silica (Geng et al, 2001), monolithic stationary phases (Okanda & El Rassi, 2006) and cellulose (Aniulyte et al, 2006). These immobilized lectins are invaluable tools for isolating and separating glycoproteins, glycolipids, polysaccharides, subcellular particles and cells. In addition, lectin affinity columns can be used to purify detergentsolubilized cell membrane components. They also are useful for assessing changes in levels or composition of surface glycoproteins during cell development and in malignant or virally transformed variants. In subsequent chapters, more detailed examples of lectin

Reversed phase chromatography is a kind of affinity interaction between a biomolecule dissolved in a solvent (mobile phase) that has some hydrophobicity (e.g. proteins, peptides, and nucleic acids) and an immobilized hydrophobic ligand (stationary phase) (Dorsey & Cooper, 1994). Reversed phase chromatography is generally more suitable for separating non-volatile molecules. The term "reversed phase" was adopted because the binding occurs between a hydrophobic ligand (octadecyl; C18) and molecules in a polar aqueous phase which is reversed from normal phase chromatography [where a hydrophilic polar ligand

In general, the macromolecules (e.g. protein or peptides) are adsorbed onto the hydrophobic surface of the column. Elution is achieved using a mobile phase which is usually a combination of water and organic solvents (such as acetonitrile or methanol) applied to the column as a gradient (e.g. starting with 95:5 aqueous:organic and gradually increasing the organic phase until the elution buffer is 5:95 aqueuos:organic). The macromolecules bind the hydrophobic surface of the column and remain until the concentration of the organic phase

When using reversed phase chromatography, the most polar macromolecules are eluted first and the most nonpolar macromolecules are eluted last: the more polar (hydrophilic) a solute is, the faster the elution and vice versa. In summary, separations in reversed phase chromatography depend on the reversible adsorption/desorption of solute molecules with

As illustrated in Figure 13, the initial step of reversed phase separation involves equilibration of the column under suitable conditions (pH, ionic strength and polarity). The polarity of the solvent can be modified by adding a solvent such as methanol or acetonitrile and an ion pairing agent such as formic acid or trifluoroacetic acid may be added. Next, sample is applied and bound to the immobilized matrix. Following this step, desorption and elution of the biomolecules is achieved by decreasing the polarity of the mobile phase (by increasing the percentage of organic modifier in the mobile phase). At the end of the separation, the mobile phase should be nearly 100% organic to ensure complete removal of all bound substances. Once everything has eluted from the column, the initial mobile phase is reapplied to the column to reequilibrate the column for a

affinity purification can be found.

subsequent sample application.

**6.7 Reversed phase chromatography**

binds to molecules in a hydrophobic nonpolar mobile phase].

is high enough to elute the macromolecules from the hydrophobic surface.

varying degrees of hydrophobicity to a hydrophobic stationary phase.

sepharose to create an affinity column. See Figure 6. This dye is capable of removing over 90% of albumin in the sample (Travis et al, 1976).

### **6.6 Lectin affinity chromatography**

Lectin affinity chromatography is one of the most powerful techniques for studying glycosylation as a protein post translational modification (Hirabayashi et al, 2002; Spiro, 2002). Lectins are carbohydrate binding proteins that contain two or more carbohydrate binding sites and can be classified into five groups according to their specificity to the monosaccharide. They exhibit the highest affinity for: mannose, galactose/Nacetylgalactosamine, N-acetylglucosamine, fucose, and N-acetylneuraminic acid (Sharon, 1998). In this affinity technique, protein is bound to an immobilized lectin through its sugar moeities (N-linked or O-linked). Once the glycosylated protein is bound to the affinity support, the unbound contaminants are washed away, and the purifed protein eluted.

Currently, many lectins are commercially available in an immobilized form. Among them, Concanavaline A (Con A) Sepharose and wheat germ agglutinin (WGA) are the most popular for glycoprotein purification. As shown in Table 3, several different types of lectin may be used in affinity chromatography.

Table 3. Some examples of lectins used for glycoprotein purification modified from current protocols in protein science.

sepharose to create an affinity column. See Figure 6. This dye is capable of removing over

Lectin affinity chromatography is one of the most powerful techniques for studying glycosylation as a protein post translational modification (Hirabayashi et al, 2002; Spiro, 2002). Lectins are carbohydrate binding proteins that contain two or more carbohydrate binding sites and can be classified into five groups according to their specificity to the monosaccharide. They exhibit the highest affinity for: mannose, galactose/Nacetylgalactosamine, N-acetylglucosamine, fucose, and N-acetylneuraminic acid (Sharon, 1998). In this affinity technique, protein is bound to an immobilized lectin through its sugar moeities (N-linked or O-linked). Once the glycosylated protein is bound to the affinity support, the unbound contaminants are washed away, and the purifed protein eluted.

Currently, many lectins are commercially available in an immobilized form. Among them, Concanavaline A (Con A) Sepharose and wheat germ agglutinin (WGA) are the most popular for glycoprotein purification. As shown in Table 3, several different types of lectin

**Elution**

0.1–0.5 M α-MeMan

0.1–0.5 M α-MeMan

GlcNAc

GalNAc

0.1–0.5 M L-Fuc or methyl-α-L-Fuc

0.1–0.5 M GalNAc

Table 3. Some examples of lectins used for glycoprotein purification modified from current

**conditions Useful for binding**

region

MeMan Similar to LCA/LCH

High-Man, hybrid, and biantennary N-linked chains

Bi- and triantennary N-linked chains with Fuc α1-6 in core

GlcNAc- and Sia- terminated chains, or clusters of *O*-GlcNAc; succinylated form selectively

binds GlcNAc>Sia

(Tn antigen)

linkages

2)Gal–

Proteins with terminal α-GalNAc or GalNAcα-*O*-Ser/Thr

Sugar chains with terminal α-Fuc, especially in α1-2 linkage, but much less with α1-3 or α1-6

Proteins with blood group A structure GalNAcα1-3(Fucα1-

**Sugar specificity**

Ca2+, Mn2+ ß-GlcNAc 0.1–0.5 M

GalNAc

90% of albumin in the sample (Travis et al, 1976).

**6.6 Lectin affinity chromatography**

may be used in affinity chromatography.

**Metal ions required**

Ca2+, Mn2+ <sup>α</sup>-Man > α-Glc

Ca2+, Mn2+ <sup>α</sup>-Man > α-Glc

*sativum;*peas) Ca2+, Mn2+ <sup>α</sup>-Man 0.1–0.5 M α-

*lunatus;* lima bean) Mn2+, Ca2+ Terminal α-

**Acronym, Organism and source**

Con A (*Canavalia ensiformis;* jack bean

LCA or LCH (*Lens culinarus;* lentil

seeds)

seeds)

germ)

snail)

HPA (*Helix promatia;* albumin gland of edible

UEA-I (*Ulex europaeus;* furze gorse seeds)

LBA (*Phaseolus*

protocols in protein science.

PSA (*Pisum*

WGA (*Triticum vulgaris;* wheat

Lectin affinity columns can be prepared by immobilizing lectins with different specificities toward oligosaccharides to a variety of matrices, including agarose (West & Goldring, 2004), silica (Geng et al, 2001), monolithic stationary phases (Okanda & El Rassi, 2006) and cellulose (Aniulyte et al, 2006). These immobilized lectins are invaluable tools for isolating and separating glycoproteins, glycolipids, polysaccharides, subcellular particles and cells. In addition, lectin affinity columns can be used to purify detergentsolubilized cell membrane components. They also are useful for assessing changes in levels or composition of surface glycoproteins during cell development and in malignant or virally transformed variants. In subsequent chapters, more detailed examples of lectin affinity purification can be found.

### **6.7 Reversed phase chromatography**

Reversed phase chromatography is a kind of affinity interaction between a biomolecule dissolved in a solvent (mobile phase) that has some hydrophobicity (e.g. proteins, peptides, and nucleic acids) and an immobilized hydrophobic ligand (stationary phase) (Dorsey & Cooper, 1994). Reversed phase chromatography is generally more suitable for separating non-volatile molecules. The term "reversed phase" was adopted because the binding occurs between a hydrophobic ligand (octadecyl; C18) and molecules in a polar aqueous phase which is reversed from normal phase chromatography [where a hydrophilic polar ligand binds to molecules in a hydrophobic nonpolar mobile phase].

In general, the macromolecules (e.g. protein or peptides) are adsorbed onto the hydrophobic surface of the column. Elution is achieved using a mobile phase which is usually a combination of water and organic solvents (such as acetonitrile or methanol) applied to the column as a gradient (e.g. starting with 95:5 aqueous:organic and gradually increasing the organic phase until the elution buffer is 5:95 aqueuos:organic). The macromolecules bind the hydrophobic surface of the column and remain until the concentration of the organic phase is high enough to elute the macromolecules from the hydrophobic surface.

When using reversed phase chromatography, the most polar macromolecules are eluted first and the most nonpolar macromolecules are eluted last: the more polar (hydrophilic) a solute is, the faster the elution and vice versa. In summary, separations in reversed phase chromatography depend on the reversible adsorption/desorption of solute molecules with varying degrees of hydrophobicity to a hydrophobic stationary phase.

As illustrated in Figure 13, the initial step of reversed phase separation involves equilibration of the column under suitable conditions (pH, ionic strength and polarity). The polarity of the solvent can be modified by adding a solvent such as methanol or acetonitrile and an ion pairing agent such as formic acid or trifluoroacetic acid may be added. Next, sample is applied and bound to the immobilized matrix. Following this step, desorption and elution of the biomolecules is achieved by decreasing the polarity of the mobile phase (by increasing the percentage of organic modifier in the mobile phase). At the end of the separation, the mobile phase should be nearly 100% organic to ensure complete removal of all bound substances. Once everything has eluted from the column, the initial mobile phase is reapplied to the column to reequilibrate the column for a subsequent sample application.

Affinity Chromatography: Principles and Applications 25

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### **7. Funds**

This work was supported by JSPS (Japan Society for Promotion of Science) Grant-in-Aid for scientific research (B) to Dr. Sameh Magdeldin (23790933) from Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in the decision to publish or in the preparation of this chapter.

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**2**

*Czech Republic*

**Affinity Interactions as**

Eva Benešová and Blanka Králová *The Institute of Chemical Technology, Prague*

 **a Tool for Protein Immobilization**

Proteins, biopolymers composed of proteinogenic amino acids, are molecules with irreplaceable functions in human organism. They have among others a role as structural components, they are involved in motion processes, they appear as significant parts of immune response and different types of protection events, they play important roles in transport and storage processes and they also occur in signalling processes. One of the most important protein functions is their role as natural biocatalysts - enzymes, because these compounds, increasing the rate of metabolic reactions, are necessary for almost all reactions in human body. The potential of enzyme application in biotechnological processes was discovered many years ago. The rapid development of molecular biology and protein engineering, enabling targeted designing of proteins with suitable features and their production in recombinant form, contributed in decisive way to the final anchoring of proteins in biotechnological practice including such areas as food industry and medicine. Also different methods of protein immobilization represent a way enabling their common and easy application in biotechnology (Demain & Vaishnav, 2009; García-Junceda et al., 2004; Murray et al., 2002; Vodrážka, 1999). A part of this improvement, focused on utilization of affinity interactions in immobilization processes, will be the major part of this

First part of this chapter will be aimed to the explanation of the importance of enzyme application in biotechnological processes and the improvement of their usage caused by immobilization. Advantages and limitations of immobilization processes will be described and some examples of their practical application in biotechnology and pharmaceutical industry will be mentioned. This part will also contain a brief summary of common

Second part of this chapter will explain the reasons for recombinant proteins preparation and the advantage of proteins modifications by techniques of molecular biology. A simple strategy for recombinant protein preparation in the simplest expression system of *E. coli* will be described for a better comprehensibility. Different methods of exploitation of affinity interactions for the protein immobilization will be referred in the last part of the chapter. The importance of achieved results for the biotechnological practice will be

**1. Introduction**

chapter.

summarized.

immobilization techniques.

## **Affinity Interactions as a Tool for Protein Immobilization**

Eva Benešová and Blanka Králová *The Institute of Chemical Technology, Prague Czech Republic*

### **1. Introduction**

28 Affinity Chromatography

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