Structural and functional repetition in a marine mussel adhesive protein.

The DOPA-rich polyphenolic protein secreted by the marine mussel Mytilus edulis establishes key chemical linkages in a water-resistant adhesive. Molecular cloning of the gene for this remarkable protein reveals its primary structure as one of the most repetitive proteins identified in the animal kingdom. Expression and purification of polyphenolic proteins from recombinant yeast have provided sufficient material to demonstrate adhesivity of these polypeptides in the laboratory. Adhesive tests reveal a water-resistant bonding capacity of the protein that is dependent on in vitro modification of tyrosine residues to DOPA and the subsequent oxidation to quinone.


Experimental and theoretical evidence for convective nutrient transport in an immobilized cell support.

Even though immobilized-cell reactors possess several engineering advantages over free-cell reactors, their full potential has not been realized because mass transfer often limits the rate of nutrient supply and product removal from immobilized cell supports. We studied the interaction between mass transfer and reaction kinetics in the anaerobic conversion of glucose to CO2 and ethanol by yeast immobilized in a porous rotating disk on the agitator shaft of a conventional CSTR. A Sherwood number correlation was used to show that external mass-transfer resistances were negligible under typical operating conditions. The modulus of Weisz based on observable reaction parameters was used to gauge the importance of pore diffusion limitations. Under conditions for which significant pore diffusion effects and hence low effectiveness factors (eta = ca. 0.1) would be predicted, the observed reaction rates were much higher than expected (eta = ca. 1), suggesting that pore diffusion limitations were at least partially relieved by convective transport of glucose into the support. Two possible mechanisms of convective transport are discussed. We hypothesize that gas evolution was responsible for the convective enhancement of glucose supply.


Antibody production in baculovirus-infected insect cells.

We have employed the baculovirus expression system for the production of a mouse monoclonal IgG antibody directed against lipoprotein I of Pseudomonas aeruginosa. Both light and heavy chain cDNAs were introduced into the baculovirus genome in a single step of homologous recombination. Insect cells that were infected with the recombinant virus stably secreted antigen-binding and glycosylated antibody molecules capable of binding the complement component C1q.


Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment.

Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment. PUF has a macroporous structure giving a high surface area to volume ratio. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 X 10(8) cells/cm3 PUF and 3.5 X 10(7) cells/cm3 PUF, respectively) in PUF stationary cultures. In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture. A maximum cell density of 2.4 X 10(7) cells/cm3 culture vessel volume was obtained in a packed-bed culture of Vero cells.


Characteristics of arachidonic acid metabolism of human endothelial cells in culture.

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E2 and thromboxane A2 in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum. After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E2 and thromboxane A2 production showed no variations during the study.


Heterohybridomas that secrete high levels of pseudomonas-specific therapeutic human monoclonal antibodies: their generation and large scale growth in an automated hollow fiber cell culture system.

Fusion of lymphoblastoid cell lines that produce human monoclonal antibodies against Pseudomonas aeruginosa with the human/mouse heteromyeloma SHM-D33 generated heterohybrids that were stable and secreted antibody in the range of 20 to 300 micrograms/ml. One of the hybridoma cell lines ws adapted to serum-free medium and maintained for 60 days in an automated hollow fiber system. During that time, 3 g of antibody was produced. Such yields make it possible to evaluate these monoclonals for their therapeutic potential in patients at risk for Pseudomonas infections.


Comparative behaviour of L-929 fibroblastic and human endothelial cells onto crosslinked protein substrates.

Studies were carried out to compare the behaviour of human umbilical vein endothelial cells (HUVEC) and L-929 fibroblastic cells towards proteins crosslinked by glutaraldehyde (GTA) or carbodiimide (CDI) proposed for coating of vascular prostheses. CDI crosslinking of bovine serum albumin used alone, or mixed with gelatin, allowed higher rates of cell growth and DNA synthesis than GTA crosslinking independent of cells. Assessment of the plating efficiency revealed a similar behaviour of both cells towards membranes and reference plastic surface in terms of percentages of bound cells. HUVEC proliferation onto CDI crosslinked gelatin and/or albumin membranes did not differ significantly whereas the growth of L-929 was enhanced onto gelatin albumin membranes in comparison with both gelatin membranes and the reference surface. The analysis of DNA synthesis corroborated the results of the growth curves and elicited a delay of the growth phases in HUVEC cultured onto CDI crosslinked membranes, unlike the L-929 fibroblast.


DNA distribution and respiratory activity of Spodoptera frugiperda populations infected with wild-type and recombinant Autographa californica nuclear polyhedrosis virus.

Spodoptera frugiperda cells were infected with a wild-type Autographa californica nuclear polyhedrosis virus and with a recombinant Autographa californica nuclear polyhedrosis virus. The recombinant virus was derived from the wild-type virus and produced beta-galactosidase instead of polyhedrin. The changes in cell size, cell growth, viability, DNA distribution, and respiratory activity were followed through the time course of the infection. The DNA content as measured by flow cytometry of infected cells increased to approximately 1.8 times the value of uninfected cells and the distributions of single-cell DNA content of the infected cells were strongly deformed. Early in the infection the respiratory activity passed through a maximum. The mitochondrial activity based on Rhodamine 123 labelling of cells infected with the recombinant virus, as determined by flow cytometry, also passed through a maximum at 24 h post infection while the mitochondrial activity of cells infected with the wild-type virus continued to increase. Evolution of single-cell mitochondrial activity was different in uninfected populations and in populations infected with wild-type and with recombinant virus. In all experiments performed, the recombinant virus influenced cell behavior and the measured parameters earlier than the wild-type virus. The influence of the multiplicity of infection was stronger for the wild-type virus than for the recombinant virus.


Waste treatment technologies from the Genesearch Group of companies.

Genesearch is a 10 year old research and development company of Australian scientists, specializing in microbiology and genetics. This research expertise has formed the basis of a number of microbial processes for waste treatment. In addition, a novel type of high yield fermenter for aerobic bacteria allows the economical production of high-potency bacterial preparations for waste treatment processes. A novel approach to the rapid biodegradation of polychlorinated biphenyls has been developed. Photochemical pretreatment partially dechlorinates the molecules, rendering them susceptible to complete and rapid digestion by wild-type soil bacteria. In the area of non-toxic waste, Genesearch has developed products for on-site treatment of, for example, grease-trap wastes and waste oil in ship bilges; and a large scale process for conversion of municipal grease wastes into protein-rich biomass. The prospects for novel biological waste treatment are improving, as public pressure grows, and as increasing government monitoring and penalties make inadequate waste disposal uneconomic.


Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction.

Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. monocytogenes.


Isolation and characterization of acetonitrile utilizing bacteria.

Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified as Chromobacterium sp. and Pseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation and P. aeruginosa grew slightly faster than Chromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (14C)acetonitrile indicated 57% of acetonitrile was degraded by Pseudomonas aeruginosa as compared to 43% by Chromobacterium. The isolates utilized different nitrile compounds as carbon substrates.


High efficiency electroporation of Lactococcus lactis subsp. lactis LM0230 with plasmid pGB301.

Electroporation-mediated transformation of Lactococcus lactis with plasmid pGB301, a 9.8 kilobase pair vector (Behnke et al. 1981), has been reported by McIntyre & Harlander (1989a). Improved transformation efficiencies of 10(2)-10(3)/micrograms DNA were achieved by altering the conditions under which the bacteria were grown prior to electroporation (McIntyre & Harlander 1989b). This present investigation sought to improve still further transformation efficiencies in order to provide a reliable high frequency transformation system for Lc. lactis subsp. lactis.


The production of recombinant beta-galactosidase in Escherichia coli in yeast extract enriched medium.

The productivity of Escherichia coli biomass and recombinant beta-galactosidase was increased in Luria broth (LB) enriched with yeast extract. In flask culture under conditions of LB limitation, yeast extract supplementation gave the highest biomass (strain HB101/pRW756) stimulation per unit of component added compared with supplementation by various amounts of amino acids, vitamins, minerals, purines/pyrimidines, tryptone, casamino acids, casein peptone or gelatin peptone. The biomass production of E. coli HB101/pRW756, XL-1 Blue/puc118, XL-1 Blue FF/puc118 and TB-1/p1034 cells was stimulated in fermentor-scale experiments with additional yeast extract in LB. Total beta-galactosidase production from plasmid genes in fermentor-scale experiments was increased 105.4% in XL-1 blue/puc118 cells, 365.5% in XL-1 blue FF/puc118 cells and 421.4% in TB-1/p1034 cells by 0.5%, 1% and 1% weight per volume of additional yeast extract in LB, respectively. Depending on different strains, the increase of the enzyme production was obtained either by increased biomass, or the combination of enhanced gene expression and increased biomass. Neither the biomass nor beta-galactosidase production was stimulated in N4830/p1034 cells by the increase in yeast extract concentration in the medium.


The D-galactose dehydrogenase gene from Pseudomonas fluorescens: characterization of mutations leading to increased expression in Escherichia coli.

The gene encoding D-galactose dehydrogenase (gld; E.C. 1.1.1.48) from Pseudomonas fluorescens is poorly expressed when cloned into Escherichia coli. Mutagenesis of the wild-type construct leads to a strong expression of gld in the heterologous host. To investigate the mutational events directing the increase in expression we constructed a gld-lacZ translational fusion which facilitated the isolation of mutants by colony screening. From several independent mutants three point mutations could be identified. They were distinguished by the sequence position of their respective single base-pair substitutions in the 5'-untranslated region of the gld gene and the degree of enhancement of enzyme activity of the gene product. The influence of these mutations on gld gene expression was analysed by S1 protection analysis which revealed that their effect was at the level of transcription.


Production and characterization of human gamma interferon from Escherichia coli.

The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.


Effect of organic substrates on biological sulphide oxidation.

Neither acetate nor higher fatty acids and glucose have a significant effect on the biotechnological process for sulphide removal at 20 degrees C, in which sulphide is oxidized to sulphur using oxygen. The oxidation of acetate and propionate with oxygen is mainly dependent on the sulphide and oxygen concentrations in the reactor. The occurrence of Thiothrix filaments in sulphide-removing waste-water treatment systems has been investigated using a fixer-film upflow reactor. The influent of this reactor consisted of anaerobically treated paper-mill waste-water, with a sulphide concentration of 140 mg/l. It was found that sulphide loading rate is the decisive parameter as to whether or not Thiothrix will develop in a sulphide-removing reactor.


Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells.

Baculoviruses are currently used as vectors for the transient high-level expression of foreign gene products in insect cells. In this study, we demonstrate that baculoviruses can also be made to continuously express a foreign gene product by using the promoter from IE1, an immediate early viral gene, to produce stably-transformed insect cells. This approach gave levels of foreign gene expression lower than those usually obtained with the lytic baculovirus expression vector system. Expression, however, was continuous and stable, and a complex human glycoprotein (tissue plasminogen activator) was processed more efficiently. We conclude that stable transformation is a feasible approach for baculovirus-mediated foreign gene expression in lepidopteran cells, particularly for products that are relatively poorly-expressed and/or processed in lytically infected cells.


A novel screening system for yeast strains capable of secreting tissue plasminogen activator.

We have developed a simple screening procedure that allowed us to identify Saccharomyces cerevisiae strains able to secrete human tissue plasminogen activator (tPA) into the culture medium. The screen can be used to isolate more efficient secretor strains and to look for novel tPA analogs. Employing one of these strains to study the effect of glycosylation on secretion, we show that glycosylation in the catalytic domain of tPA plays an important role in folding and/or secretion of the molecule. Removing this glycosylation site resulted in a 3-5-fold reduction in the level of tPA secretion. We anticipate that this system will prove useful in studying yeast secretory pathway as well as structure-function relationships in the tPA molecule.


Selective flocculation with chitosan in Escherichia coli disintegrates: effects of pH and nuclease treatment.

The flocculation of cell debris from a beta-galactosidase constitutive E. coli with chitosan as a flocculant was studied to investigate the possibility of obtaining a selective flocculation in cell disintegrates with high product recoveries. The flocculation removed 98% of the cell debris by 30 min sedimentation under gravity, which should be compared to a separation of the cell debris without flocculation of only 70% by centrifugation at 15,000 g. Optimal flocculation dosages varied between 12 and 43 mg chitosan g-1 dry weight of cells, depending on pH. The yield of the product beta-galactosidase reached 60% at optimal pH. Hydrolysis of the nucleic acids by DNAase and RNAase decreased the optimal flocculation dosages considerably. The study showed that the flocculation is somewhat selective, since chitosan also removed 85% of the nucleic acids and 50% of the proteins, which contributed to the purification of the protein solution.


A bifunctional vector system for controlled expression and subsequent release of the cloned gene product by phi X174 lysis protein-E.

A new bifunctional Escherichia coli cloning vector, pSB50, is presented. The plasmid allows controlled expression of a gene of interest under control of the lac promoter and the subsequent release of the cloned product by the use of bacteriophage phi X174 lysis protein-E, the gene of which is under control of the phage Lambda pL promoter. To ensure optimal repression of the Lambda pL promoter and the lac promoter in plasmid pSB50, E. coli strain UB89-1 was constructed which carries a chromosomal copy of the lambda cl857 repressor allele and the laclq1 allele, respectively. Here, we employ the E-based lysis system to release human prourokinase to the culture medium.


Synthesis and biological activity of some 5-substituted aminomethyl-8-hydroxyquinoline-7-sulphonic acids.

5-Aryl (or alkyl)-8-hydroxyquinoline-7-sulphonic acids have been prepared by the Mannich reaction of 8-hydroxyquinoline-7-sulphonic acid with primary and secondary amines. Their bactericidal activities have been determined.


Amperometric bi-enzyme based biosensor for the detection of lactose--characterization and application.

Based on the glucose oxidase-beta-galactosidase sequence an enzyme probe for the specific determination of lactose has been developed. beta-Galactosidases from different sources have been compared, the sensor containing beta-galactosidase from Curvularia inaequalis has been characterized in respect of optimal pH, enzyme loading, apparent activity and functional stability. The response of the bi-enzyme probe depends linearly on lactose concentration between 0.02 and 3.00 mmol dm-3. The application to different milk and foodstuff samples resulted in good correlations toward enzymatic photometric (y = (0.956x-1.67) mmol dm-3) and infrared detection (y = (1.0772x-0.3909)%). Using a measuring frequency of 100 h-1 the serial imprecision is about 2% for diluted milk, urine, or foodstuff samples.


Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.


Use of a rapid bioluminescence assay for detecting cyanobacterial microcystin toxicity.

The recent rise in the awareness of the occurrence of toxic cyanobacterial blooms in aquatic environments, with associated human health problems and animal deaths, has increased the need for rapid, reliable and sensitive methods of determining cyanobacterial toxicity. A luminescent bacterial toxicity test was assessed as a complement to the established mouse bioassay. Seventeen samples including pure cyanobacterial microcystin-LR hepatotoxin, laboratory isolates and natural blooms of cyanobacteria were tested and toxicity data compared with mouse LD50 values. Microcystin-LR and all five microcystin-containing cyanobacterial samples, hepatotoxic by mouse test gave EC50 values of less than 0.46 mg/ml in bioluminescence-based Microtox assays. Of 11 samples non-toxic by mouse bioassay, only two gave an EC50 of less than 0.98 mg/ml by bioluminescence assay. It is suggested that the Microtox bioluminescence assay may prove useful in the preliminary screening of cyanobacterial blooms for microcystin-based toxicity.


Penicillin production by glucose-derepressed mutants of Penicillium chrysogenum.

Wild-type strains of Penicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.


Improvement of glidobactin A production by Polyangium brachysporum.

Glidobactins A, B, and C are lipopeptide antitumor antibiotics produced by the gliding bacterium Polyangium brachysporum sp. nov. No. K481-B101. The production of glidobactin A was examined in shake flasks and laboratory fermentors. Medium screening and optimization led to approximately five fold increases in glidobactin A titers in shake flasks and a ten fold increase in titers in 40-l batch fermentations. Utilization of a stepped glucose feeding protocol resulted in glidobactin A titers of 1860 micrograms/ml after 144 h of fermentation.


A method for the stabilisation of recombinant plasmids in yeast.

The stability of a yeast plasmid can be improved using deliberately induced cyclic changes in the dissolved oxygen tension (DOT), during continuous culture in a non-selective, undefined medium. The resultant stability of the plasmid under DOT cycled conditions is strongly dependent on the growth rate of the culture, with complete stabilisation at lower growth rates. We propose a mechanism for the stabilisation and suggest that the method can be applied to other recombinant yeast strains.


Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.

Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.


Regulation of biosynthesis of bacilysin by Bacillus subtilis.

Production of the dipeptide antibiotic bacilysin by Bacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose. Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin. Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down. Ammonium salts were poor for bacilysin production when used as the sole nitrogen source. When added to the standard medium containing glutamate, they suppressed antibiotic production. Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source. No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate. When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth. Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations. Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components. Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6 x 10(-4) M.


In vitro expression of Lac-PTS and tagatose 1,6-bisphosphate aldolase genes from Lactococcus lactis subsp. cremoris plasmid pDI-21.

A 4.4-kb EcoR1-EcoR1 DNA fragment from the Lactococcus lactis subsp. cremoris plasmid pDI-21 encoded the tagatose 1,6-bisphosphate (TBP) aldolase gene and the Lac-PTS genes. In vitro transcription-translation using Escherichia coli S30 extract showed the synthesis of 41,000-, 23,000- and 12,000-dalton proteins which correspond to the TBP-aldolase, Lac-PTS enzyme II, and factor III proteins respectively.


The role of monic acid A in pseudomonic acid A biosynthesis in Pseudomonas fluorescens.

The putative role of monic acid A as a biosynthetic intermediate of the antibiotic pseudomonic acid A, providing a C17 moiety requiring only esterification with a C9 fatty acid, has been tested by administration of [14C]monic acid A early in the pseudomonic acid A idiophase of a Pseudomonas fluorescens fermentation. [14C]Monic acid A was not taken up by the cells and the pseudomonic acid A subsequently accumulated was not radiolabelled. Experimental demonstration of the biosynthetic role of monic acid A and the potential use of monate analogues in biotransformations will require unexpectedly elaborate strategies to ensure the uptake of these compounds into bacterial protoplasts. The impermeability to monic acid A explains for the first time why it is not an antibiotic.


Cosolvent assisted protein refolding.

The use of cosolvents in aqueous systems has been shown to enhance protein refolding and decrease aggregation. In this study, we have used polyethylene glycol (PEG) in the molecular weight range of 1000 to 8000 Daltons to effectively increase the rate of refolding and prevent aggregation of the model protein, bovine carbonic anhydrase B (CAB). At concentrations of 3 and 30 g/l, PEG increased the rate of recovery of active protein in the absence of aggregation. Using 3 g/l PEG (3350 MW), the refolding rate was three fold greater than the observed normal refolding rate. The observed rate enhancement was caused by PEG acting on the first intermediate in the CAB refolding pathway to increase the rate of formation of the second intermediate. The interaction of PEG with the first intermediate also prevented its self-association during refolding and at equilibrium. The stabilization of this first intermediate resulted in complete recovery of active protein under normal aggregating conditions.


Production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859.

5-Methyluridine was produced continuously from thymine and inosine by immobilized enzymes, which consisted of thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase obtained from Bacillus stearothermophilus JTS 859. The process was carried out in a column reactor at 60 degrees C for 17 d without any bacterial contamination under non-aseptical conditions. Half-lives of the activity of the immobilized enzymes were 47 d and 4.5 d at 60 degrees C and 70 degrees C, respectively, although half-life of the crude enzyme was only 14 h at 70 degrees C.


Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis.

Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(alpha)-phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine.


Enzyme-linked immunosorbent assays for detection of Pseudomonas fluorescens in sediment samples.

A strain-specific antibody to Pseudomonas fluorescens was used to develop two enzyme-linked immunosorbent assays: a sandwich and a competitive assay. Both assay types could be used to perform rapid and sensitive detection of the target organism in extracts of sediment samples.


A 6 h microslide immunodiffusion assay for confirmed detection of staphylococcal enterotoxins.

Use of appropriately balanced immunological reagents and incubation of microslides at 45 degrees C resulted in confirmed assay results in 6 h for staphylococcal enterotoxins. The sensitivity of the 45 degrees C-6 h assay was comparable to the 37 degrees C-24 h assay: 0.1 micrograms of staphylococcal enterotoxin A/ml.


Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines.

Recombinant Autographa california baculoviruses expressing genes for pseudorabies virus glycoprotein (gp50T), human plasminogen (HPg), and beta-galactosidase (beta-gal) were used to infect 23 cell lines or strains. The objectives were to compare amounts of recombinant proteins expressed in the cell lines, compare yields from clones and parent lines, investigate the effects of long-term culture in serum-free medium on production, and determine if some lines yield gp50T with different glycosylation patterns. For HPg, IZD-MB0503 had the highest yield and four other lines (IPLB-TN-R2, IPLB-SF-1254, IPLB-LdEIta, and CM-1) had levels above that of SF-9 cells. For gp50T, four lines (IPLB-HvT1, IPLB-SF21AE, IPLB-SF21AE-15, and IPLB-SF-1254) had higher amounts than SF-9 cells. Some lines yielded gp50T with molecular mass about 1000 daltons larger than that from SF-9 cells, which suggests increased oligosaccharide processing. Equally high levels of beta-gal were expressed in three lines (SF-9, IZD-MB0503, and BCIRL-PX2-HNV3). The major conclusion is that no single cell line produced highest yields for all three recombinant proteins. Four lines were cultured in serum-free medium for 31-34 passages and then infected with the three recombinant viruses. For most cell line-recombinant combinations, the yields in serum-free medium were equal to or better than those in serum-supplemented medium. Medium composition had a much stronger effect on foreign gene expression than on susceptibility of cells to wild-type virus.


Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene.

The expression in Aspergillus is described of genes, coding for intracellular and extracellular proteins controlled by the promoter region of the constitutively and efficiently expressed glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) of Aspergillus nidulans. Both the homologous gpdA and the heterologous Escherichia coli beta-galactosidase (lacZ) and beta-glucuronidase (uidA) genes could be expressed intracellularly at levels as high as 10-25% of total soluble protein. Efficient extracellular production of A. niger glucoamylase could be achieved with a fusion-gene containing the region of the glucoamylase gene coding for the mature protein preceded by a synthetic fungal signal sequence. Extracellular production of a heterologous protein, E. coli beta-glucuronidase, with such a fusion was much less efficient. Only very low levels of beta-glucuronidase were detected in the culture fluid, whereas considerable enzyme activity was detected in the mycelium.


Molecular characterization and functional analysis in Aspergillus nidulans of the 5'-region of the Penicillium chrysogenum isopenicillin N synthetase gene.

The isopenicillin N synthetase gene (pcbC) was isolated from a genomic library of Penicillium chrysogenum BC39813, a penicillin production strain. The nucleotide sequence, including 555 bp upstream of the translation start site was determined. Various deletions within the pcbC 5'-region were constructed and linked to the Escherichia coli lacZ gene. An Aspergillus nidulans argB strain was transformed with DNA of these constructions. The region essential for promoter function could be localized between positions -307 and -89 by analyzing beta-galactosidase expression of transformants containing a single copy of the corresponding plasmid integrated at the homologous argB locus. A region responsible for regulatory effects concerning nitrogen metabolism was identified by determining beta-galactosidase activities in cell-lysates of transformants cultivated under varying growth conditions. Two major transcription start sites at positions -131 and -132, as well as a further upstream located site at position -397 +/- 1 could be located by primer extension studies employing RNA isolated from P. chrysogenum BC39813.


Strain improvement of Penicillium chrysogenum by recombinant DNA techniques.

The penDE gene from Penicillium chrysogenum has been isolated; the gene is located in close vicinity of the pcbC gene. Amplification of the pcbC-penDE gene cluster in Penicillium chrysogenum Wis54-1255 leads to a significant increase in penicillin production. In selected transformants an increase of up to 40% is observed.


Design of two immobilized cell catalysts by entrapment on gelatin: internal diffusion aspects.

Experimental results obtained during the design of two immobilized cell catalysts by entrapment on gelatin are presented. Strong diffusional limitations are found and explained with the usual parameters and models, introducing an empirical correlation between substrate concentration and effectiveness factor. The effect of particle size, enzyme load, and specific activity in the system is discussed in terms of cooperation between bioengineers and geneticists.


Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.


Feedback regulation and the intracellular protein profile of Streptomyces griseus in a cycloheximide fermentation.

Two-dimensional gel electrophoresis (2-D PAGE) was used to study the intracellular protein profile of Streptomyces griseus in relation to cycloheximide (CH) biosynthesis. Four proteins (CR1-CR4) were found to be significantly and specifically repressed by addition of the antibiotic (1 g/l at 72 h) to a producing fermentation. Synthesis of these proteins was specific to the idiophase, concurrent with CH production. Initial addition of CH to the production medium resulted in slightly lower synthesis rates of two of the proteins (CR1 and CR2), while significantly delaying the onset of synthesis of the other two (CR3 and CR4). Finally, neutral polymeric resin was added to the fermentation to alleviate feedback regulation of CH synthesis, giving roughly a twofold increase in the antibiotic production rate. Production of proteins CR3 and CR4 was increased approximately tenfold immediately following resin addition, but returned to the control rate of synthesis after 24 h.


Production of recombinant human erythropoietin in Bowes melanoma cells in suspension culture.

Recombinant DNA technology was used to insert a fetal liver genomic library ApaI fragment encoding for human erythropoietin (Epo) into Bowes melanoma cells. The cells expressed the erythropoietin gene, and Epo was secreted into the culture medium together with the normally-secreted tissue plasminogen activator. Attempts to grow the cells in glass spinners in Dulbecco's medium supplemented with fetal bovine serum produced cell aggregates growing in suspension. When calcium-free suspension culture media (Joklik, DME-S, McCoy 5A-S) were used, single cell suspension cultures were obtained and high Epo production observed. When attempts were made to scale up the small glass spinners, poor growth or Epo production occurred unless the vessels were aerated. This was shown to be because of the drop in pH, possibly due to CO2 accumulation, rather than due to oxygen depletion. It was shown that a semi-continuous operation could be achieved in aerated 8-1 spinners fitted with either a conventional stirrer or a vibromixer agitator. The system was scaled up to a 100-1 stainless steel vessel fitted with a vibromixer agitator. This system was operated for over 4 months with weekly harvests producing over 100 million units of Epo in about 1000-1 of culture fluid. Interference by the serum proteins with downstream purification of the hormone from the culture fluid made the use of serum-free media highly desirable. Studies showed that the Epo was produced in serum-free systems containing peptones.


Redesigning t-PA for improved thrombolytic therapy.

Attempts are being made to redesign the structure of tissue-type plasminogen activator (t-PA) in order to increase its plasma half-life, increase its fibrin affinity or decrease its rate of interaction with plasma inhibitors. The principal strategies employed so far have been to construct hybrid enzymes, to mutate the polypeptide sequence of t-PA or to add extra fibrin-binding elements. It has been relatively easy to alter the half-life of t-PA but more difficult to do this with retention of the full specific activity of the molecule; the most promising molecules will have to be evaluated in the clinic before we know whether the redesign of t-PA has been truly successful.


Stability of a host-vector system based on complementation of an essential gene in Escherichia coli.

Antibiotic selection is the most common selection system for plasmid-containing bacteria. This technique, nevertheless, can be a source of problems during the expression of heterologous genes in Escherichia coli. We have developed an alternative selection system based on the complementation of a chromosomal auxotrophic (dapD2) mutation by the corresponding wild type gene carried on a plasmid. We show that the system effectively selects for the presence of plasmid on solid and liquid medium. In addition, we have observed a loss of viability associated with high levels of gene expression and accumulation of a heterologous protein, but the selective power and improved intrinsic stability of the dap+ plasmid, compared to a beta-lactamase (bla) based vector, excludes overgrowth of the culture by plasmidless cells.


A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.


Temperature and induction effects on the degradation rate of an abnormal beta-galactosidase in Escherichia coli.

Intracellular protein degradation was investigated using an unstable fragment of Escherichia coli beta-galactosidase, the CSH11 mutant, as a model protein. This abnormal protein was expressed from a single copy gene in the chromosome and is converted to a detectable degradable intermediate. The in vivo degradation rates of both beta-galactosidase fragments were measured using pulse-chase radioactive labeling techniques, and their intracellular concentrations were determined using alpha-complementation assays. In the physiological range of 30 to 37 degrees C, the apparent degradation rate constant for the CSH11 fragment follows Arrhenius behavior; while the intermediate's apparent degradation rate constant is nearly unchanged. However, above 37 degrees C the degradation rates of both fragments increase significantly. Analysis of the labeled intermediate's rate of change above 40 degrees C reveals that the CSH11 fragment is being degraded by a second pathway which does not produce the intermediate. When the induction level of the abnormal beta-galactosidase was varied the degradation rates of both fragments behaved similarly, but they unexpectedly decreased with increasing IPTG concentration. The two parallel degradation pathways for CSH11 apparently operated at only the lower IPTG levels. The measured degradation rates did not correlate directly with the intracellular concentration of abnormal proteins.


Decreased production of para-hydroxypenicillin V in penicillin V fermentations.

Penicillin V (phenoxymethyl penicillin) is produced by industrial strains of Penicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain of P. chrysogenum produces an undesirable penicillin byproduct, para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, via para-hydroxylation of POAc and subsequent incorporation of the p-OH phenoxyacetic acid into the penicillin molecule. Most of the p-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level of p-OH penicillin V produced by the control strain ranges up to 10-15% of the total penicillins produced. 3-Phenoxypropionic acid and p-bromophenylacetic acid partially inhibit the formation of p-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels of p-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which produce p-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.


Beijerinckia indica var. penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity.

Beijerinckia indica var. penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, gamma-irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.


Alginate biosynthesis in mucoid recombinants of Pseudomonas aeruginosa overproducing GDP-mannose dehydrogenase.

The Pseudomonas aeruginosa algD gene, encoding GDP-mannose dehydrogenase (GMD) and cloned at Chakrabarty's Laboratory in the expression vector pMMB24 (plasmid pVD211), was mobilized into P.aeruginosa strains 8821 and 8821M. Strain 8821M was a high-alginate-producing variant, spontaneously obtained from mucoid strain 8821, with derepressed levels of GMD, a key enzyme in the regulation of alginate biosynthesis, leading to the irreversible oxidation of GDP-mannose to GDP-mannuronic acid. A slight increase in the level of GMD, in both strains harboring the plasmid pVD211 and batch-grown at 37 degrees C without IPTG induction, led to the increase of production rate and the final concentration of alginate produced by control strains harboring the cloning vector. However, the viscosity of the aqueous solutions prepared with the alginate (3 g l-1) produced by mucoid strains harboring pVD211 was lower than those with the alginate produced by the controls (shear rates in the range 0.6-12 s-1). The specific activity of GMD assayed in crude extracts from cells harboring pVD211 and subjected to IPTG induction (0.5 and 3 mM) presented the highest values. However, either the rate of biosynthesis and final concentration of alginate or the viscosity of solutions prepared with the alginate produced by recombinants grown with IPTG were lower than that possible without overproduction. Therefore, the stimulation of the alginate pathway only by manipulating the rate of the step catalysed by GMD, although possible within certain levels, was at the expense of the final exopolysaccharide quality.


Extractive purification of enzymes from animal tissue using aqueous two phase systems: pilot scale studies.

Pilot scale trials have been carried out to assess the feasibility of using PEG-salt aqueous phase systems for extraction and purification of enzymes from animal tissue on an industrial scale. Comminuted bovine liver was used as a starting material, and it was easy to separate a clear upper phase containing proteins of interest from a mixture containing 20% biomass, 15% PEG and 8% phosphate using a disc separator. Similar attempts with decanters were unsuccessful. Second-phase separation was simply accomplished by the addition of salt to the separated, clear upper layer and standing or allowing passage through a disc separator. The method was tested using continuous mixing on the GBF continuous mixing aqueous phase extraction plant, with and without computer control. Good separations were achieved. The enzyme superoxide dismutase was purified using this method yielding a 4-fold purification factor with respect to soluble protein and a recovery rate of 83%, with the enzyme in a clarified solution suitable for further processing by chromatographic methods. The general applicability of this method, its economics and its potential application in industry are discussed.


Transport and kinetics in sandwiched membrane bioreactors.

A bioreactor in which living yeast cells are sandwiched between an ultrafiltration membrane and a reverse osmosis membrane was constructed, and experiments were performed for the conversion of substrate glucose to product ethanol. A set of equations that include both transport through a series of barrier layers and bioreaction rate were developed to predict the performance of the sandwich bioreactor. The above equations were solved by using numerical values for the transport parameter and the bioreaction rate constant, and the results are compared with the experimental data.


Saccharomyces cerevisiae strains that overexpress heterologous proteins.

We describe a system that facilitates the selection of host mutants that overproduce a range of secreted and internally produced heterologous proteins in Saccharomyces cerevisiae. These mutants were initially selected for their ability to oversecrete recombinant human albumin (rHA), as detected by a direct visual assay that relies upon antibody precipitation in solid media. Yeast strains that were able to synthesize and secrete increased levels of rHA also produced elevated levels of internally expressed proteins including alpha 1-antitrypsin Pittsburgh variant and plasminogen activator inhibitor type 2.


Antibody immobilization onto glow discharge treated polymers.

Previous studies have shown that certain glow discharge treated polymers strongly retain adsorbed albumin and fibrinogen. On the basis of this phenomenon, we have investigated the possibility of immobilizing antibodies on glow discharge treated surfaces for diagnostic immunoassay applications. As a model for antibody immobilization, bovine IgG was immobilized on the following polymers: polyethylene (PE), tetrafluoroethylene glow discharge treated PE (TFE/PE), poly(ethylene terephthalate) (PET), TFE/PET, poly(tetrafluoroethylene) (PTFE), ethylene glow discharge treated PET (E/PET) and hexamethyldisiloxane glow discharge treated PET (HMDS/PET). IgG was radiolabeled with 125I and immobilized by either of the following two methods: (a) physical adsorption of IgG on untreated and glow discharge treated polymers or (b) physical adsorption of albumin followed by chemical coupling of IgG to albumin by glutaraldehyde. IgG concentration as well as adsorption times were varied in order both to optimize the immobilization conditions and to investigate the adsorption and retention mechanisms. To evaluate the efficiency of the immobilization techniques, blood plasma, Tween-20, and sodium dodecyl sulfate (SDS) were used to elute the adsorbed IgG layer. We found that IgG was successfully immobilized on the fluorocarbon glow discharge treated surfaces by using either the physical adsorption or the glutaraldehyde coupling method, although the former is more efficient than the latter method.


Ambruticin S production in air-lift and stirred-tank bioreactors.

Using the method of equi-inocular synchronized comparative fermentation (EISCF) the cultivation of Sorangium cellulosum So ce 10 and production of the polyketide antibiotic ambruticin S was compared in stirred-tank and air-lift reactors of different geometry. This method requires that inocula originate from the same pre-culture and cultivation parameters are synchronized to similar values. Similar ambruticin yields were obtained from both reactor systems provided that the concentration of dissolved oxygen (DO) was maintained above a certain value (ca. 40%). For cultivation of S. cellulosum it is the DO level rather than the oxygen transfer rate which presents the proper criterion for scale-up and comparative reactor studies.


Immobilization of Escherichia coli JM103[pUC8] in kappa-carrageenan coupled with recombinant protein release by in situ cell membrane permeabilization.

Immobilization of Escherichia coli JM103[pUC8] was carried out with kappa-carrageenan as the support matrix. Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells. As compared to suspension cultures, much higher beta-lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid-bearing cells (improved plasmid stability) were observed in immobilized cell cultures. Further enhancement in excretion of the recombinant protein (beta-lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA). While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA. Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes. Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of beta-lactamase when compared to the enzyme production in EDTA-free cultures. More frequent addition of EDTA increased the period of retention of plasmid-bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of beta-lactamase. Improvement in plasmid stability in EDTA-treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid-free and plasmid-bearing cells, since plasmid-free cells were subject to more reduction in specific growth rate than were plasmid-bearing cells.


The genetic stability of Penicillium chrysogenum transformants in a fermentor.

A number of transformants of Penicillium chrysogenum have been obtained with the plasmid vector p3SR2. Southern analysis showed that transformation had occurred by integration of vector sequences into the nuclear DNA of the fungus. A number of transformants were tested for stability of the transformed phenotype in agar medium and some were found to be unstable. Two transformants, shown to be stable in agar culture, were grown in 5-1 batch fermentors for further stability tests. Over periods of up to 312 h in the fermentor both transformants were 100% stable with respect to the transformed phenotype. In addition Southern analysis of DNA extracted from the spent mycelium showed that no change had occurred in the position of the integrated vector sequences within the transformant nuclear DNA.


Reversion of L-lysine inhibition of penicillin G biosynthesis by 6-oxopiperidine-2-carboxylic acid in Penicillium chrysogenum PQ-96.

6-Oxopiperidine-2-carboxylic acid (OCA; cyclic alpha-aminoadipic acid) reverses the L-lysine inhibition of penicillin G production by Penicillium chrysogenum PQ-96. The reaction probably depends on the recovery of L-alpha-aminoadipic acid for penicillin G production from OCA.


A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability.

A cell-associated pullulanase (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55,000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr- Ala- Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75-80 degrees C and at 80-85 degrees C, respectively. The enzyme was stable up to 90 degrees C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae----B. acidopullulyticus----B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.


Cloning and amplification of the gene encoding an extracellular beta-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates.

We have cloned and determined the nucleotide sequence of the gene encoding an extracellular beta-glucosidase (bgl1) from the cellulolytic fungus Trichoderma reesei. The predicted open reading frame of the bgl1 gene is interrupted by two putative introns of 70 and 64 bp and encodes a protein with a calculated molecular weight of 75,341. The genomic segment encoding bgl1 was cloned into a vector that contained the selectable marker gene, amdS. Transformation of T. reesei with this vector resulted in several stable transformant strains all possessing an increased copy number of the bgl1 gene integrated into the genome together with elevated rates of glucose production from avicel. One transformant produced an extracellular cellulase with a five-fold increase in the rate of production of glucose from cellobiose, a 33% rate increase from avicel, and a 17% increase from phosphoric acid swollen cellulose. These data suggest that the cellulolytic activity of T. reesei strains may be specifically improved by transformation with cloned cellulase genes.


Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response.

The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences. Comparison of the tac, trp and lambda PL promoters showed that expression was maximal under tac control. Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression. The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions. In E. coli DG75 grown at 37 degrees C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction. Growth at 30 degrees C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times. Western blot analysis of the products showed that degradation of rscuPA was much larger at 37 degrees C than at 30 degrees C. Using E. coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures. Expression in this strain was even more efficient than in E. coli JM101 carrying the lon mutation. It is concluded that induction of the general heat-shock response in E. coli must be avoided to obtain stabilization of rscuPA. This drastically improves the overall yield of rscuPA from recombinant E. coli strains.


Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture.

Recombinant human interferon-gamma production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.


Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli.

The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, L-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain. About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.


Heterologous gene expression in continuous cultures of budding yeast.

In a previous paper we have studied the expression of beta-galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity. In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector. The maximal level of expression is influenced by the dilution rate. Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation. A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements.


"Differentiation induction" culture of human leukemic myeloid cells stimulates high production of macrophage differentiation inducing factor.

A suitable procedure for the production of human monokines was defined as 'differentiation-induction' culture. Human monocytic leukemia THP-1 cells were well-differentiated from nonfunctional promonocytes into macrophage-like cells by the induction with a combination of mezerein, retinoic acid, and a Mycoplasma fermentans extract. The differentiated THP-1 cells secreted a high amount of macrophage differentiation-inducing factor (DIF) activity and concomitantly produced other known monokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta), into the medium. These results suggest that other novel human monokines may also be found in the conditioned medium of THP-1 cells induced by the 'differentiation-induction' culture conditions defined in this study. Macrophage DIF was purified to homogeneity and NH2-terminal amino acid sequence analysis revealed that macrophage DIF is very similar or identical to human leukemia inhibitory factor (LIF). The cDNA encoding human LIF was isolated using the polymerase chain reaction, and a clone producing 3.7 micrograms/10(6) cells day recombinant LIF was selected from Chinese hamster ovary (CHO) cells which were transfected with the LIF cDNA. The recombinant LIF production in CHO cells was quantified using MTT reduction assay with M1 cells.


Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein.

Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.


Elucidation and optimization of the medium constituents controlling antibiotic production by the cyanobacterium Nostoc muscorum.

A study has been made to determine which nutrient factors control antibiotic production by the cyanobacterium, Nostoc muscorum. A two-phase approach was employed using a factorial method to explore the response surface and a steepest ascent method to climb the response surface to the region of the optimum. It was found that nitrate and iron were the factors significantly affecting antibiotic production; 26.4 mM nitrate and 6 microM iron were the optimal concentrations for maximizing antibiotic production by N. muscorum.


Production processes of recombinant IL-1 beta from Bacillus subtilis: comparison between intracellular and exocellular expression.

The human IL-1 beta coding sequence derived from a cDNA library was inserted into two different plasmid expression vectors, pSM214 and pSM308, which promote the synthesis of recombinant products intracellularly and exocellularly, respectively. The hybrid constructs pSM261 and pSM320 were obtained. Bacillus subtilis SMS118 was transformed with these plasmids and the recombinant strains were grown in 1 litre bioreactors. Different growth conditions were analyzed to optimize yields both in terms of biomass and IL-1 beta production. In the pSM261-harbouring strain, IL-1 beta was synthesized in the cytoplasm to levels ranging from 1 to 2.7 mg g-1 of cells, corresponding to up to 40 mg l-1 of the culture. In contrast, SMS118(pSM320) was able to secrete 0.27 mg of natural IL-1 beta per g of cells (6.7 mg l-1 of culture). Processes for the purification of IL-1 beta from the supernatant and the biomass of the two cultures were also developed and compared in terms of yield and simplicity of the purification schemes. From our data it turns out that the route of intracellular expression is very efficient and superior to the one which results in secretion of IL-1 beta. This indicates that the use of B. subtilis as a recombinant host in biotechnology is not strictly dependent on its ability to secrete proteins into the culture medium.


Expression of the Bacillus subtilis levanase gene in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for the inulin hydrolyzing enzyme levanase which was previously cloned from Bacillus subtilis was fused to the tac-promoter. Overexpression in Escherichia coli resulted in high amounts of intracellularly produced levanase (up to 20 U mg-1). After removal of the bacterial 5' sequences, the levanase gene was also cloned into a yeast expression vector based on the PGK-promoter. Clones containing the intact levanase gene including the bacterial signal sequence gave rise to synthesis of active levanase by Saccharomyces cerevisiae transformants. A considerable amount of levanase protein was found in the culture medium (around 0.5 U ml-1) indicating efficient secretion of B. subtilis levanase from yeast.


Dried gel hybridization in place of southern hybridization for detection of Listeria monocytogenes DNA fragments.

A simple, sensitive, dried gel DNA hybridization method for detection of Listeria monocytogenes DNA fragments is described. DNA samples were fractionated on an agarose gel. The gel was then denatured in NaOH-NaCl and neutralized in Tris-NaCl. The resulting agarose gel was dried and hybridized with 32P-labelled DNA probe. No transfer to nitrocellulose membranes was used.


Isolation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine from culture broths by covalent chromatography.

An efficient procedure for the isolation of reduced delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) from culture supernatants of beta-lactam antibiotic-producing microorganisms is described. The method utilises covalent chromatography to isolate thiols from culture broths that have been deproteinised and undergone borohydride reduction. 2-Pyridyl disulphide activated thiopropyl Sepharose was employed batchwise to isolate the thiols present in such broths from cultures of the known ACV excreter Cephalosporium acremonium N2 and the penicillin producer Penicillium chrysogenum P2. ACV was separated from these mixtures of thiols by gel permeation chromatography. Reversed-phase HPLC analysis showed the ACV to be of a high purity unless isolated from a highly complex culture medium.


Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis.

The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-poly-acrylamide gel electrophoresis characterizations. The NH2-terminal sequence analysis of the secreted PME revealed two different NH2-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and alpha-amylase signal peptide.


Reshaping a human monoclonal antibody to inhibit human respiratory syncytial virus infection in vivo.

We transferred the complementarity determining regions from a murine monoclonal antibody that neutralizes infection by respiratory syncytial virus (RSV) to a human IgG1 monoclonal antibody. The resulting reshaped human antibody lost affinity for RSV, but an additional alteration to one of the framework regions restored binding affinity and specificity. This second generation reshaped human monoclonal antibody cross-reacted with all clinical isolates of RSV tested and both prevented disease and cured mice even when administered four days after infection. We expect the antibody will prove useful in the management of this major childhood disease.


Continuous regeneration of NAD(H) covalently bound to a cysteine genetically engineered into glucose dehydrogenase.

We introduced a cysteine residue on the surface of glucose dehydrogenase from Bacillus subtilis using site-directed mutagenesis. To this mutant, an NAD-analogue was covalently attached by a disulphide bridge so that it was active intramolecularly. The glucose dehydrogenase-cys44-NAD complex, which contained one reactive NAD molecule per subunit of glucose dehydrogenase, was operated together with lactate dehydrogenase in a coupled enzymatic regeneration of NAD(H) in a hollow fiber reactor. L-lactate and gluconic acid were continuously produced from pyruvate and D-glucose, respectively, with a turnover number of 45 cycles per minute for each NAD molecule. The total turnover per coenzyme was 135,000 for the first 2.5 days.


Broad host-range vector for efficient expression of foreign genes in gram-negative bacteria.

A broad host-range expression plasmid was constructed comprising the incQ replicon, the recA promoter from Escherichia coli and the g10-L ribosome binding site (RBS) derived from bacteriophage T7. The structural genes for porcine somatotropin (pst) and E. coli beta-galactosidase (lacZ) were used to monitor gene expression in a diverse collection of Gram-negative bacterial hosts: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas testosteroni, Serratia marcescens and Erwinia herbicola. The E. coli recA promoter was functional in this wide range of hosts and was inducible by the addition of nalidixic acid. Moreover, the level of lacZ expression was often at least as high as that observed in E. coli. Previous studies had shown that the g10-L RBS was superior to a simple "consensus" RBS sequence for expression of foreign genes in E. coli. Here we demonstrate a 38 to 70 fold increase in expression in two Pseudomonas hosts using the g10-L RBS, indicating that the translational enhancer present in the g10-L RBS is also functional in other bacteria. The juxtaposition of these transcriptional and translational elements in a broad host-range vector provides a simple way to evaluate alternate hosts for recombinant protein production.


High level expression of human tissue-type plasminogen activator gene in mouse C127 cell.

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.


On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.


Molecular cloning in Lactobacillus helveticus by plasmid pSA3::pVA797 co-integrate formation and conjugal transfer.

A gene encoding beta-glucanase activity from Bacillus amyloliquefaciens was subcloned in both orientations into plasmid shuttle vector pSA3. In only one orientation could a co-integrate be generated with the conjugative plasmid pVA797. The plasmid co-integrate was conjugated into Lactobacillus helveticus strain CNRZ450, where it was stably maintained without antibiotic selection and exhibited beta-glucanase activity. This method of introducing cloned DNA into thermophilic lactobacilli will facilitate the study of heterologous gene expression in non-transformable species.


Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil.

Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O2 consumption and CO2 evolution) and survival during a 14-day incubation period at 20 degrees C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O2 consumption/CO2 evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.


Screening of staphylococci for the toxic shock syndrome toxin-1 (TSST-1) gene.

Dot blot hybridization was used to screen 820 staphylococci for the presence of the gene coding for TSST-1. The DNA of 33 strains among 70 Staph. aureus strains isolated from suspected toxic shock syndrome (TSS) cases hybridized with the probe. These results agreed perfectly with those obtained with a phenotypic method (immunodiffusion). Among 608 Staph. aureus strains isolated over a period of one month from hospitalized patients, 66 (11%) hybridized with the probe; of these strains, 64 (97%) were found to produce TSST-1 in vitro. None of 145 coagulase-negative staphylococcal strains harboured DNA hybridizing with the probe. The data indicate that this genotypic assay is suitable for epidemiological studies.


Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression.

We report the first successful production of transgenic goats that express a heterologous protein in their milk. The production of a glycosylation variant of human tPA (LAtPA--longer acting tissue plasminogen activator) from an expression vector containing the murine whey acid promoter (WAP) operatively linked to the cDNA of a modified version of human tPA was examined in transgenic dairy goats. Two transgenic goats were identified from 29 animals born. The first animal, a female, was mated and allowed to carry the pregnancy to term. Milk was obtained upon parturition and was shown to contain enzymatically active LAtPA at a concentration of 3 micrograms/ml.


Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and characterization of the recombinant enzyme.

A glycosylation variant of human tissue-type plasminogen activator (tPA) designated longer-acting tissue-type plasminogen activator (LAtPA) was extensively purified from the milk of a transgenic goat by a combination of acid fractionation, hydrophobic interaction chromatography and immunoaffinity chromatography. This scheme provided greater than 8,000-fold purification of the protein, a cumulative yield of 25% and purity greater than 98% as judged by SDS gel electrophoresis. SDS gel electrophoresis revealed that the transgenic enzyme was predominantly the "two chain" form of the protease. The specific activity of the purified transgenic protein, based on the average of the values obtained for three different preparations, was 610,000 U/mg as judged by amidolytic activity assay. This was approximately 84% of the value observed for the recombinant enzyme produced in mouse C127 cells. Analysis of the transgenic protein indicated that it had a significantly different carbohydrate composition from the recombinant enzyme produced in C127 cells. Molecular size analysis of the oligosaccharides from the transgenic and C127 cell-derived LAtPA preparations confirmed their differences and showed that the mouse cell-derived preparation contained larger, complex-type N-linked oligosaccharide structures than the material produced in goat mammary tissue.


Cloning of the nitrate-nitrite reductase gene cluster of Penicillium chrysogenum and use of the niaD gene as a homologous selection marker.

A new homologous transformation system for the filamentous fungus Penicillium chrysogenum is described. The system is based on complementation of niaD mutants using the nitrate reductase structural gene (niaD) of P. chrysogenum. Spontaneous niaD mutants were identified after selection for chlorate resistance, in growth tests and subsequent complementation with the niaD gene of Aspergillus oryzae. The P. chrysogenum niaD gene was isolated from a genomic library using the Aspergillus nidulans niaD gene as a probe. After subcloning of the hybridizing fragment, the vector obtained, pPC1-1, was capable of transforming a P. chrysogenum niaD mutant at an average of 40 transformants per micrograms of circular DNA. Southern analysis of genomic DNA from a number of transformants showed that pPC1-1 DNA was integrated predominantly at sites other than the niaD locus. Using hybridization analysis it was shown that the niaD gene of P. chrysogenum is clustered with the nitrite reductase gene (niiA). From analysis of the nucleotide sequences of parts of the niaD and niiA genes of P. chrysogenum and comparison of these sequences with nucleotide sequences of the corresponding A. nidulans genes it was deduced that the P. chrysogenum genes are divergently transcribed.


Use of the urokinase-type plasminogen activator gene as a general tool to monitor expression in transgenic animals: study of the tissue-specificity of the murine whey acidic protein (WAP) expression signals.

Urokinase-type plasminogen activator (uPA) is a proteolytic enzyme able to convert the zymogen plasminogen into the strong protease plasmin. The availability of very sensitive tests to measure the enzymatic activity of a plasminogen activator renders the corresponding gene an ideal candidate for the detection of promoter activity. In this paper we describe the utilization of the human uPA gene as detector of tissue-specificity of the murine whey acidic protein (WAP) expression signals in transgenic mice. The WAP promoter has been previously investigated for the production of foreign proteins in the milk of transgenic animals. In our genetic constructions, the human uPA cDNA was linked to the promoter region as well as to 3'-end distal sequences of the WAP gene. Five transgenic lines were obtained in which, however, expression levels of human uPA in the milk were still quite low. Surprisingly, four of these five positive transgenic mice show a consistent activity of the WAP promoter in brain extracts compared to other tissues.


Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine alpha s1-casein.

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with alpha s1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the amino acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of alpha s1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the alpha s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No clear consensus sequence of amino acid residues surrounding the cleavage sites could be identified.


Growth and protein production kinetics of a murine myeloma cell line transfected with the human growth hormone gene.

A model mammalian cell system for the production of recombinant proteins was investigated. Murine myeloma cells which had lost the ability to produce both heavy and light chain immunoglobulin molecules were transfected with a vector containing the immunoglobulin heavy chain promoter and enhancer elements linked to the human growth hormone gene. The growth kinetics of G32, a clonal isolate, were found to be similar to both the parent myeloma and hybridomas. However, production of hGH by G32 was growth associated, rather than as a secondary metabolite as is the case for hybridomas. In addition, G32 produced hGH at molar levels greater than most hybridomas.


Single chain antibody variable regions.

The use of antibodies or antibody fragments for targeting tumors (either for tumor imaging or as carriers for drugs or toxins), has encountered problems of clearance, and non-specific or inefficient binding in clinical trials. A novel approach, linking two antibody variable fragments (Fvs), with a short peptide to generate a continuous polypeptide chain, may be able to overcome some of these problems. Since these single chain antibody variable regions (scFvs), are transcribed from constructed 'genes', large-scale production in, for example, E. coli, should be straightforward.


Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs.

The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3'-azido-3'-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mM) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent Km and Vmax revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest.


Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine beta-casein.

The action of the cell-envelope proteinase (PIII-type) from Lactococcus lactis ssp. cremoris AM1 on bovine beta-casein was studied. The results were compared with those obtained earlier with (PI-type) proteinases from the cell envelope of other L. lactis strains. From a 4-h digest (pH 6.2; 15 degrees C) of beta-casein made with the PIII-type proteinase, 24 peptides were isolated and purified by selective precipitation followed by semi-preparative reversed-phase HPLC. Altogether, these peptides accounted for the preferential splitting of 16 peptide bonds in beta-casein by the PIII-type proteinase. In nine cases the primary cleavage site (P1-P'1) was a Glx-X or X-Glx peptide bond. In ten cases at least one large hydrophobic residue (Met, Leu, Tyr, Phe) formed part of the cleavable bond. The P2-P3 and/or P'2-P'3 regions of the substrate consisted of hydrophobic and/or negatively charged side chains or of side chains potentially involved in hydrogen bonds. Nine of the peptide bonds split were reported previously to be also susceptible to cleavage by PI-type proteinases, although the kinetics may be different. The PIII-type proteinase shows a broader specificity in its initial cleavage of beta-casein than does the PI-type.


Continuous production of tissue plasminogen activator (t-PA) by human embryonic lung diploid fibroblast, IMR-90 cells, using a ceramic bed reactor.

Ceramic pieces composed of 99.5% Al2O3, 3 to 6 mm long, were found to be a good matrix for growth of the human embryonic lung diploid fibroblast, IMR-90 cells. The tissue plasminogen activator (t-PA) was secreted in DME medium containing proteose peptone as a t-PA inducer. In addition, production of t-PA was enhanced by increasing extracellular CaCl2, from 3.6 to 5.4 mM. In order to eliminate negative feed-back control caused by t-PA produced and thus raise productivity, perfusion cultivation was performed using a ceramic-packed bed column, with a recirculating vessel. The recirculating vessel was used to mix fresh medium with spent medium, and to control dissolved oxygen concentrations in the extracellular environment by stirring. In continuous production using the packed bed column with 2 kg of ceramics (phi = H = 150 mm), increasing dilution rate to 0.5 day-1 could reduce product inhibition at 3-4 x 10(5) cells/ml. Cellular productivity of 560 IU/10(6) cells/day was obtained over 40 days and corresponded to the volumetric productivity of 183 IU/ml/day.


The kinetics of plasmid loss.

Instability of bacterial cloning vectors can present a serious problem when direct selection for plasmid-encoded phenotypes is undesirable, ineffective or impractical. Antibiotic selection may provide a satisfactory solution in enclosed fermentors but not where recombinant organisms are part of complex microbial consortia after release into the outside environment. In the past decade there has been significant progress towards understanding the causes of plasmid loss and the lessons learned from these studies can be used in the design of a new generation of stable vectors.


Strategies for improving plasmid stability in genetically modified bacteria in bioreactors.

Exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins is often hampered by the problem of plasmid instability. A wide range of strategies have been reported for improving the stability of recombinant organisms. A combination of manipulating both the genetic design of recombinants and the conditions of culturing the organisms may be used to achieve stable host-vector associations during culture of recombinant organisms in bioreactors.


The nasal delivery of peptides and proteins.

Many drugs of the future will be therapeutically active peptides and proteins developed through recombinant-DNA technology. A major factor limiting their exploitation is the current lack of appropriate non-parenteral delivery systems. Nasal systems incorporating absorption enhancers may provide a convenient, efficient means of administering protein and peptide therapeutics.


Protein A insensitive ELISA detection of staphylococcal enterotoxin B.

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin B was developed using rat monoclonal antibodies as capture antibodies and as a biotinylated conjugate. This test was sensitive, less than 1 ng/ml of enterotoxin B was detected and interference by protein A was prevented by the use of rat monoclonal antibodies of the IgG2a isotype which were insensitive to protein A even at concentrations greater than 1000 ng/ml.


Monitoring of the production of monoclonal antibodies by hybridomas. Part I: Long-term cultivation in hollow fibre bioreactors using serum-free medium.

The long-term cultivation of hybridoma cells in hollow fibre bioreactors using serum-free medium, was monitored with respect to quantitative and qualitative aspects of the produced mAbs, cell viability, LDH and proteolytic activity. During the culture periods of hybridoma cells producing mAb OT-1C and 3A, the mAb concentration showed a decreasing trend with a concomitant increase of IgG fragments. The major IgG fragments did not bind the antigen and the molecular weights were significantly different from the corresponding IgG heavy and light chains. In addition, a good correlation was found between cell lysis, the presence of acid protease(s) and IgG fragments. The physicochemical and immunochemical properties of the "intact" mAbs (such as molecular weights, IEF patterns and affinity) did not change significantly during the culture period.


Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.


Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion.

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crossings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.


Production of functional IgM Fab fragments by Saccharomyces cerevisiae.

The aim of this study was to express and secrete functional mouse IgM fragments in yeast. The heavy chain cDNA was truncated at two different sites, yielding genes coding for the complete VH domain. In one of the truncated genes, the CH1 domain is complete, while in the other gene 18 bp are missing from the 3' terminus of the CH1 region. Both shortened genes were coexpressed in Saccharomyces cerevisiae with a cDNA gene encoding a full length mouse Ig light chain. We show that only the longer form of the truncated heavy chain together with the light chain produced and secreted functional IgM Fab fragments.


IgG production in hybridoma batch culture: kinetics of IgG mRNA, cytoplasmic-, secreted- and membrane-bound antibody levels.

During batch cultivation of the I.13.17. hybridoma cell line, there is a 15 to 20-fold decrease in the levels of cytoplasmic and membrane-bound mAb, and a 7 to 10-fold decrease in the cellular levels of kappa and gamma chain mRNAs, as the cells pass from the exponential into the decline phase of growth. The profile of the specific mAb production rate does not correlate with the kinetics of either the cytoplasmic mAb or the specific mRNAs throughout the culture. Flow cytometry analyses have revealed that dead cells, which account for 40 to 70% of total cells during the decline phase, might significantly interfere with the determination of cytoplasmic mAb levels of cell-lysates ELISA and with the calculation of the specific mAb production rate. Possible influences of these parameters on mAb synthesis and secretion during hybridoma batch culture are discussed.