Reaction rates for the production of selected hormones by solid-phase synthesis.

Reaction rates have been measured for the production of selected bradykinins, angiotensins, and endorphins by solid-phase synthesis. Reactor liquid concentrations are monitored by using a UV detector and flow cell. Experimentation has been limited to low excesses of the t-Boc amino symmetrical anhydride. More than 500 attachments have been monitored. Most data obtained from resins with 1% cross-linking show second-order behavior with reaction rate constants between 0.5 and 8 L/(mol.s). The reaction rate is affected by chemical structures of both the attached amino symmetrical anhydride and the anchored amino terminus on the peptide fragment. Increasing reaction temperature and initial solution concentrations promotes reaction rate. The structure of the polymer support affects not only the reaction rate but also the observed reaction kinetics.


Solvent selection strategies for extractive biocatalysis.

This report follows the development of systematic solvent screening strategies for the identification of superior pure solvents and introduces techniques for the identification of effective coextractants. Specifically, methods to predict the biocompatibility and extractant capability of solvents are discussed. Biocompatibility is predicted by using heuristic data or the correlations between bioactivity and the logarithm of the partition coefficient of the solvent or the concentration of solvent in the cell membrane. A computer program, known as the extractant screening program or ESP, has been developed to effectively predict the behavior of virtually any product in any solvent/aqueous system. It is demonstrated that a biocompatible yet poor solvent can be mixed with a toxic solvent that has better extractant properties to yield a mixture with improved solvent characteristics that is still biocompatible. The fact that solvents do not mix in an ideal manner is exploited by using ESP to identify solvent mixtures that are still biocompatible at relatively high concentrations of toxic solvent.


Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.


Activities of Candida rugosa lipase and other esterolytic enzymes coated on glass beads and suspended in substrate and water vapor: enzymes in thin liquid films.

Candida rugosa lipase was enzymatically active when coated on glass beads and exposed to mixtures of substrate and water vapor over a range of relative humidities up to 100%. Evidence was obtained for operation of the enzyme in a thin liquid film of concentrated buffer on the surface of the glass beads. Formation of the thin film was associated with hygroscopicity of the buffer used to suspend the enzyme in preparation of the enzyme-coated beads. At some buffer concentrations estimated to be on the bead surface, the enzyme was partially soluble and both soluble and insoluble forms were enzymatically active. The vapor mode of operation over a range of relative humidities had comparatively small effects on kinetic constants for hydrolysis of ethyl acetate, which were also similar to those in phosphate buffer. The extent of reaction occurred in the order hydrolysis greater than alcoholysis greater than ester interchange greater than esterification. Reaction preference between alcoholysis and hydrolysis changed as acyl chain length of substrate increased with C. rugosa lipase, as well as with Rhizopus arrhizus lipase and porcine liver esterase, with details depending on the enzyme. The vapor mode approach has the potential of being used with a wide variety of substrates, as shown by the ability to obtain hydrolysis at 30 degrees C with substrate vapor pressures as low as 0.08 mm Hg and with substrates with boiling points as high as 206 degrees C.


Microscopic visualization of insect cell-bubble interactions. I: Rising bubbles, air-medium interface, and the foam layer.

Through the use of microscopic, high-speed video technology, the interactions of two suspended insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with air and oxygen bubbles were studied. Events such as cell-bubble attachment, cell-bubble collision, cell transport into the foam layer, and trapping of cells in the foam layer are presented and discussed. Based on these observations and those in a companion paper (Chalmers, J. J.; Bavarian, F. Biotechnol. Prog. 1991, following paper in this issue) and the experimental and theoretical work of other researchers, several mechanisms of cell damage as a result of sparging are presented.


Stabilization of enzymes by their specific antibodies.

In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.


Microscopic visualization of insect cell-bubble interactions. II: The bubble film and bubble rupture.

In this paper, the second in the series, the use of a microscopic, high-speed video system to study the interactions of two suspended insect cells strains, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with rupturing bubbles is reported. Events such as the adsorption of cells onto the bubble film and the mechanism of bubble rupture were observed. On the basis of these observations and the experimental and theoretical work of other researchers on bubble rupture and cell death as a result of sparging, it is proposed that cells are killed by the rapid acceleration of the bubble film after rupture and the high levels of shear stress in the boundary layer flow associated with bubble jet formation.


Automatic bioprocess control. 1. A general concept.

Automation of bioprocesses is presented and discussed. A general concept is applied to laboratory scale reactors as well as to large scale production facilities consisting of many unit operations with a hierarchical and highly modular structure. The implementation of non-dedicated and intelligent analytical subsystems is foreseen. Hard- and software requirements are discussed in view of the functional requirements of both scientific research and production engineering. Some practical experience is reported using several different components in parallel installations.


Elution conditions and degradation mechanisms in long-term immunoadsorbent use.

The limited life of immunoadsorbents used for the large-scale purification of biological macromolecules poses a significant limitation to the more widespread application of this technology. In this study, the binding activity of a monoclonal antibody (MAb) to bovine serum albumin (BSA) was measured as a function of pH, ionic strength, and varying concentrations of KSCN, ethylene glycol, or DMSO. Low pH (2.5) and 3 M KSCN each reduced the antibody binding constant below 6 x 10(5) L/mol, meeting criteria derived from a simple chromatographic model for identifying effective eluents. A panel of six MAb to BSA was exposed repeatedly to adsorption conditions and the two eluents. Four MAb lost less than 50% of their initial binding capacity over 100 cycles. The other two lost 75% of their initial capacity. One MAb was stable when exposed to low pH but lost binding capacity with KSCN. In all cases, the equilibrium constant was unchanged. The loss of capacity was also shown to be a strong function of antibody loading: at 14.5 mg/mL, 98% of the initial binding capacity of one MAb was lost within 40 cycles, versus 75% loss at 1 mg/mL. Antibody leakage and nonspecific adsorption of contaminants were not responsible for significant loss of antibody activity over time.


Theoretical analysis of the effect of cell recycling on recombinant cell fermentation processes.

A cell recycle system is studied for two-stage continuous fermentation. Cell recycle around the second stage provides higher cell concentrations than processes without recycle and a longer residence time of the cell, which is necessary for inducible products, especially in recombinant cell fermentation. Residence time distribution of the cell in the fermentor is important for the optimization of inducible products. The residence time distributions are studied for the cases with and without significant cell growth in the second stage. With cell growth in the second stage, three cases are considered. These are the cases of (1) zero residence time for two daughter cells after the cell division, (2) zero residence time of one daughter cell after the cell division and inherited residence time for the other daughter cell from the mother cell after the cell division, and (3) two daughter cells having the residence time of the mother cell after the cell division.


A new method for estimating population doubling time of vertebrate cells.

Based upon the fact that the amount of DNA doubles after each cycle, a technique was developed in which the population doubling time for cell cultures was determined from the ratio of its DNA content to [3H]thymidine incorporation under conditions in which cell numbers cannot be readily determined. This technique may be useful in determining what proportion the sample is of the total cell population.


Propagation of recombinant vaccinia virus in HeLa cells: adsorption kinetics and replication in batch cultures.

The influence of various culture parameters on infection and replication of recombinant vaccinia virus in HeLa cells was examined during various phases of viral replication. A modified form of the model of Valentine and Allison (Biochim. Biophys. Acta 1960, 40, 393-399) model was used to predict successfully the viral adsorption rates in cell suspensions. An experimentally determined aggregation factor, epsilon, was included in the model to account for deviations of the observed adsorption rates from those predicted by the earlier model. It was also shown that the ionic strength, ionic species, and serum proteins present in the medium significantly altered the adsorption kinetics of the virus. The lysosomotropic base chloroquine was found to enhance viral infection more than 2-fold during the penetration step of viral infection. It was also demonstrated that cells infected during the exponential growth phase gave higher viral yields than those infected during the lag or stationary growth phases and the initial viral MOI did not significantly alter viral yields. Finally, it was demonstrated that viral infection of HeLa cells grown in 4-L bioreactor batch cultures resulted in increased death and glucose uptake rates and significantly lower growth rates.


Stability of mutant type II dihydrofolate reductase proteins in suppressor strains.

In the course of study of a (Glu-58 to Gln-58) mutant type II dihydrofolate reductase (DHFR), it was found that the altered DHFR was poorly produced in vivo. Investigations with several common laboratory Escherichia coli strains including htpR and lon strains bearing plasmids expressing the Gln-58 DHFR indicated a correlation of rapid degradation with the presence of a sup+ phenotype. The supo strain MC1061(p3) was transformed with a series of plasmids containing the Gln-58 DHFR gene with and without an additional supF gene, and expression levels were compared. The supF+ constructs exhibited little accumulation of the Gln-58 DHFR, while reasonable levels were found in the supo cases. Experiments with extracts of plasmid-free sup+ and supo strains showed rapid degradation by certain strains compared to MC1061(p3) and this degradation was not dependent upon ATP. In another route to increasing the stability of labile DHFR derivatives, mutagenesis of a strain bearing a N-terminally shortened Gln-58 DHFR was performed. Selection and analysis of a trimethoprim-resistant stable mutant showed that this DHFR gene contained a triple repeat of leu-pro-ser in the enzymatically non-essential N-terminal portion of the protein.


Bioseparation using affinity techniques.

Bioseparation of proteins from dilute solutions using different novel affinity procedures is reviewed. Emphasis is also placed on the quality of the product separated. Whenever possible, physical insights into the separation procedure are provided, besides indicating suitable directions where appropriate further research may be carried out. The procedures analyzed are different affinity chromatographic techniques, affinity separation using liquid perfluorocarbon supports, water soluble nonionic surfactants for affinity bioseparations, affinity cross-flow filtration, bioaffinity separation using reversed micelles, affinity precipitation and dual-functional affinity protein purification.


Solasodine production from self-immobilised Solanum aviculare cells.

Procedures were developed for 'self-immobilisation' of Solanum aviculare cells to eliminate the need for artificial immobilisation supports. Depending on the cytokinin used in liquid medium, compact aggregates 0.4-2.0 cm in diameter were formed without dispersed cells also being present. Histochemical analysis showed that the aggregates were structurally organised to facilitate nutrient transport. Growth, sugar uptake and solasodine production were measured in shake-flask cultures. Most of the product was stored in the aggregates to reach a maximum concentration of 0.3% dry weight; this is between 1.5 and 10 times the levels reported for suspended cells under similar conditions. A substantial amount of solasodine was produced after growth ceased. The maximum rate of solasodine production was about 0.22 mg g-1 d-1. A simple air-driven bioreactor was tested for culture of the aggregates; solasodine yields were comparable to those measured in shake flasks.


Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization.

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.


Single chain antibody (SCA) encoding genes: one-step construction and expression in eukaryotic cells.

We report the expression, in eukaryotic cells, of a gene encoding a single chain antibody (SCA) and a rapid method for the construction of such genes. A SCA directed against the aromatic dye fluorescein was synthesized from a gene constructed by means of the simultaneous use of four PCR primers and templates of both light and heavy chain immunoglobulin cDNAs in the form of either plasmid clones or reverse transcribed hybridoma RNA. Two of the primers were partially complementary to one another and encoded the polypeptide linker which joins the immunoglobulin light and heavy chain variable domains of the SCA polypeptide. A functional, hapten-binding product was synthesized from the gene thus constructed in both E. coli and the fission yeast, Schizosaccharomyces pombe. Our results demonstrate that gene constructs encoding single chain antigen binding proteins can be synthesized very rapidly with only limited sequence information about the pertinent light and heavy chain immunoglobulin genes, and, that neither murine codon usage bias, Thermus aquaticus DNA polymerase infidelity, nor the eukaryotic cellular environment preclude the synthesis of functional single chain antigen binding proteins in non-lymphatic, non-murine eukaryotic cells.


Heritable damage to yeast caused by transformation.

The introduction of plasmid DNA into yeast by transformation or electroporation, but not by cytoduction, results in the induction of a slow growth phenotype. This phenotype is inherited as a dominant Mendelian trait, which is only exhibited in the absence of the native 2 mu nuclear DNA plasmid of yeast. The use of recombinant DNA technology in yeast, therefore, does not necessarily manipulate the genome in a precise and completely defined way.


Non-animal alternative toxicity tests for detergents: genuine replacements or mere prescreens?

Non-animal toxicity tests and testing strategies for use in identifying the potential toxic hazard of chemicals and products, and in providing information for use in risk and safety assessment, are in the course of development, validation and evaluation. Possible replacement alternatives to the rabbit Draize eye irritancy test are discussed, and results obtained for two in-vitro tests developed by FRAME (the kenacid blue test and the neutral red release test), applied to 19 surfactants and 32 formulations, are discussed. It is concluded that there are now legal and moral requirements that relevant and reliable non-animal tests should be developed and accepted for use whenever possible.


Primary structure analysis of polypeptides.

Primary structure determination together with other techniques such as amino acid analysis, peptide mapping, and polyacrylamide gel electrophoresis provide a physical characterisation of a polypeptide. This has been valuable in the research field for some time, but is now being applied to problems which arise in the areas of process development, formulation and QA of protein products. Probably the most significant contribution is elucidation of processing events, since this may go undetected by other techniques. Thus, with the emergence of various protein products from biotechnology, a technique which has been of great value in research activities for many years is finding its place in the range of techniques used for process development, formulation and QA.


Efficient production of human alpha-amylase by a Bacillus brevis mutant.

A cDNA for mature human salivary alpha-amylase was directly joined to a sequence encoding the signal peptide of the middle wall protein (MWP) gene of Bacillus brevis 47. This hybrid gene was placed downstream from the multiple promoter region of the MWP gene on a low copy-number plasmid vector, pHW1. B. brevis 47 carrying the plasmid produced 0.9 mg/l of active human alpha-amylase in the medium. A B. brevis 47 mutant obtained on mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine produced an increased amount of the alpha-amylase (6 mg/l). When the fused gene was inserted into a high copy-number expression vector, pNU200, and then introduced into the mutant, a large amount (60 mg/l) of the alpha-amylase was produced in the medium. The alpha-amylase showed approximately the same specific activity and molecular weight as those of the natural enzyme. The mutant showed higher sensitivity to various antibiotics than the original strain, and altered cell wall and cytoplasmic membrane protein compositions. The results of reversion analysis suggested that a single mutation is responsible for the above phenotypes and hyper-productivity of human alpha-amylase.


Effect of soil/contaminant interactions on the biodegradation of naphthalene in flooded soil under denitrifying conditions.

The mineralization of 14C-labelled naphthalene was studied in pristine and oil-contaminated soil slurry (30% solids) under denitrifying conditions using a range of concentrations from below to above the aqueous phase saturation level. Results from sorption-desorption experiments indicated that naphthalene desorption was highly irreversible and decreased with an increase in the soil organic content, thus influencing the availability for microbial consumption. Under denitrifying conditions, the mineralization of naphthalene to CO2 occurred in parallel with the consumption of nitrate and an increase in pH from 7.0 to 8.6. When the initial substrate concentration was 50 ppm (i.e. close to the aqueous phase saturation level), about 90% of the total naphthalene was mineralized within 50 days, and a maximum mineralization rate of 1.3 ppm day-1 was achieved after a lag period of approx. 18 days. When added at concentrations higher than the aqueous phase saturation level (200 and 500 ppm), similar mineralization rates (1.8 ppm day-1) occurred until about 50 ppm of the naphthalene was mineralized. After that the mineralization rates decreased logarithmically to a minimum of 0.24 ppm day-1 for the rest of the 160 days of the experiments. For both of these higher concentrations, the reaction kinetics were independent of the concentration, indicating that desorption of the substrate governs the mineralization rate. Other results indicated that pre-exposure of soil to oil contamination did not improve the degradation rates nor reduce the lag periods. This study clearly shows the potential of denitrifying conditions for the biodegradation of low molecular weight PAHs.


A hybrid bioreactor for high density cultivation of plant cell suspensions.

A hybrid bioreactor was developed for the production of secondary metabolites from high density cultivation of plant cell suspensions. Some of the advantages of both air-lift and cell-lift by agitation were combined. The addition of a decanting column also made it possible to run a perfusion system for high density culture or to run a two-stage culture efficiently. Cell growth and the production of berberine from Thalictrum rugosum in the hybrid bioreactor are reported in this paper. A cell density up to 31 g/l was obtained by perfusion without any problems in mixing or loss of cell viability and the specific berberine productivity was comparable to that in shake flasks. The maximum berberine concentration was 88 mg/l at 3 weeks of operation and declined thereafter.


Long-term cultivation of anchorage-independent animal cells immobilized within reticulated biomass support particles in a circulating bed fermentor.

Long-term cultivation of anchorage-independent animal cells immobilized within porous biomass support particles (BSPs) using a gas-stirred circulating bed fermentor (CBF) was investigated. Inoculation of mouse myeloma MPC-11 (ATCC CCL 167) cells into reticulated polyvinyl formal resin BSPs (3 x 3 x 3 mm; mean pore diameter, 60 microns; porosity, 0.88) and the repeated batch culture of inoculated cells were performed under gentle circulation of BSPs, induced by sparging air from the base of the fermentor. The glucose uptake rate of cells decreased in the initial period just after the start of circulation, since a relatively large number of cells leaked from the BSPs. After that period, the uptake rate gradually increased and the leakage of cells diminished. In the meantime, when inoculated cells were incubated statically by introducing air into days before circulating the BSPs, glucose consumption became very rapid and cell density in the BSPs reached at least 10(7) cells/cm3 BSP. Thus, a long-term cultivation without significant leakage of cells and with high cell density in BSPs was successfully achieved in the CBF-BSP system.


Cultivation of anchorage-dependent animal cells in microsphere-induced aggregate culture.

Diethylaminoethyl-derivatized dextran microspheres were used to cultivate Chinese hamster ovary, 293, Vero and swine testicular cells. Cells became attached to the microspheres but did not spread out. Instead, they grew in a more spherical shape and eventually formed multiple-cell-layer aggregates. Viability in these aggregates remained high after the cultures reached high cell concentrations. This cultivation method allows a high cell density to be achieved with a low microsphere concentration.


Genetic analysis of the phosphinothricin-tripeptide biosynthetic pathway of Streptomyces viridochromogenes Tü494.

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricyl-alanyl-alanine (Ptt). Ptt-non-producing mutants were isolated following N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or UV light treatment of spore suspensions. In co-synthesis and bioconversion experiments the mutational blocks in the biosynthetic pathway were localized. The mutant NTG1 was analysed in detail. This mutant acts as a secretor for all other mutants. From bioconversion experiments with presumptive precursors circumstantial evidence was obtained that NTG1 is mutated in a gene involved in the alanylation of N-acetyl-demethyl-phosphinothricin. Using a cosmid gene library the DNA region complementing the defective gene of mutant NTG1 was isolated on a 4-kb BamHI fragment. Subcloning experiments showed that a 3-kb BglII/BamHI fragment is sufficient for complementation of mutant NTG1.


Construction and physiological characterization of glyceraldehyde-3-phosphate dehydrogenase overproducing transformants of Aspergillus nidulans.

The construction and characterization of glyceraldehyde-3-phosphate-dehydrogenase (GPD) overproducing transformants of Aspergillus nidulans and their behaviour in acetate-limited continuous cultures and glucose-grown batch cultures are described. The A. nidulans acetamidase deletion strain MH1277 was transformed with the homologous gpdA gene on a vector with the homologous acetamidase-gene (amdS) as a selection marker. Transformant A1 contains about nine integrated copies of the gpdA gene, and shows a proportional gene-dosage GPD production of about 22% of the total soluble cell protein. Compared to the wild-type MH1277, A1 has higher growth yields and reaches higher specific growth rates on both acetate and glucose, which could be due to the key position of GPD in glycolysis and gluconeogenesis.


Calcium inhibition of efrotomycin production by Nocardia lactamdurans.

Efrotomycin is a modified polyketide antibiotic of the elfamycin family that has use in the area of pig husbandry. Optimization of the fermentation medium for production of efrotomycin by Nocardia lactamdurans revealed that the fermentation is sensitive to hard water and certain lots of cottonseed flour used to prepare a complex fermentation medium. A limited metal ion analysis of the hard water indicated that calcium ions are present at concentrations found to be inhibitory by the addition of calcium chloride to medium prepared with distilled water. Similarly, a correlation between lots of cottonseed flour that poorly supported the fermentation and high calcium levels is presented. Further experimentation revealed that by altering the sterilization conditions of the cottonseed flour, the inhibitory effect of poor lots could be prevented.


Structural analysis of Bacillus licheniformis 86 surfactant.

A tentative structure and composition of a surfactant, BL-86, produced by Bacillus licheniformis 86 is described. The surfactant is a mixture of lipopeptides with the major components ranging in size from 979 to 1091 Da and varying in increments of 14 Da. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portion of the surfactant. There are 7 amino acids per molecule. The peptide portion is composed of the following amino acids: glutamic acid or glutamine (glx), aspartic acid or asparagine (asx), valine, leucine, and isoleucine at a ratio of 1.0:1.0:1.4:3.0:0.6, respectively. The leucine is present as both the D and L isomers at a ratio of about 2:1, respectively. Forty percent of the molecules contain L-valine instead of L-isoleucine. The glx and asx are present as a combination of L-glutamic acid and L-asparagine and/or L-glutamine and L-aspartic acid. The N-terminus of the peptide is blocked, most likely by a peptide bond to the lipid portion. An ester carbonyl structure is present, which could be a part of a lactone ring connecting the beta position of the lipid to one of the carbonyl groups in the peptide. The lipid portion is composed of, on average, 8-9 methylene groups, and contains a mixture of linear and branched tails. Results of DCI-MS and FAB-MS analyses, as well as surface tension measurements, of purified BL-86 HPLC fractions support the proposed composition.


Molecular biology of human muscle disease.

The molecular revolution that is transforming the entire biomedical field has had far-reaching impact in its application to inherited human muscle disease. The gene for Duchenne muscular dystrophy was one of the first cloned without knowledge of the defective protein product. This success was based upon the availability of key chromosomal aberrations that provided molecular landmarks for the disease locus. Subsequent discoveries regarding the mode of expression for this gene, the structure and localization of its protein product dystrophin, and molecular diagnosis of affected and carrier individuals constitute a paradigm for investigation of human genetics. Finding the gene for myotonic muscular dystrophy is requiring the brute force approach of cloning several million bases of DNA, identifying expressed sequences, and characterizing candidate genes. The gene that causes hypertrophic cardiomyopathy has been found serendipitously to be one of the genetic markers on chromosome 14, the beta myosin heavy chain.


Production of human interleukin-3 using industrial microorganisms.

We expressed a cDNA encoding the multicolony stimulating factor interleukin-3 in a variety of cell types, including bacteria, yeast and mammalian cells. After evaluation of the advantages and disadvantages of each potential system, we designed a production and purification scheme using Bacillus licheniformis. The purification consists of hydrophobic interaction chromatography, two steps of ion exchange chromatography and gel filtration. The purified and formulated product entered clinical trials in November 1989.


Synthesis of wild type and mutant human hemoglobins in Saccharomyces cerevisiae.

We have expressed human alpha and beta-globin cDNA clones from separate, synthetic galactose-regulated hybrid promoters contained on a single plasmid in Saccharomyces cerevisiae. Co-expression of the alpha and beta-globin chains in S. cerevisiae results in the assembly of these proteins into soluble tetrameric hemoglobin that accumulates to 3-5 percent of the total cell protein. Endogenously produced heme is incorporated into the tetramer and the protein produced is functionally and structurally indistinguishable from human Ao hemoglobin. This expression system has been used to produce both wild type hemoglobin and a low O2-affinity hemoglobin mutant that has oxygen binding and dissociation characteristics similar to human whole blood. The yeast expression system we describe may be suitable for the production of a recombinant hemoglobin based blood substitute as well as for detailed structure-activity studies of human hemoglobin.


High level expression of the humanized monoclonal antibody Campath-1H in Chinese hamster ovary cells.

We have cloned the light and heavy chain cDNAs for the humanized monoclonal antibody Campath-1H and expressed them in Chinese hamster ovary (CHO) cells using a dihydrofolate reductase (dhfr) amplification procedure. Each cDNA was positioned under control of the strong human beta-actin promoter/polyadenylation signals and used to evaluate alternative vector design and amplification procedures. By employing a dual selection co-transfection strategy, initial transformants accumulated antibody levels of 0.5 micrograms/ml after 4 days continuous culture. When subjected to successive rounds of selection in medium containing stepwise increments in methotrexate (MTX), stable cell lines were obtained that secreted up to 200 micrograms/ml of Campath-1H during the same period. This reflects a productivity of 100 micrograms/10(6) cells/day and demonstrates the potential of engineering CHO cells for the production of recombinant antibodies.


Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and cell sorting.

The carotenoid pigment astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) is an important component in feeds of aquacultural animals. It is produced as a secondary metabolite by the yeast Phaffia rhodozyma, and the isolation of rare mutants that produce increased quantities is limited by the lack of genetic selections. As a model system for enriching mutants increased in production of secondary metabolites, we have used quantitative flow cytometry/cell sorting (FCCS) to isolate astaxanthin hyperproducing mutants of the yeast. Experimental conditions were developed that gave a quantitative correlation of fluorescence and carotenoid content. In mutated populations, a 10,000-fold enrichment of carotenoid-overproducing yeasts was obtained. Distinctive differences were detected by FCCS in fluorescence and forward scatter values of mutant and wild-type populations of yeasts. Comparison of wild-type and mutant clones by fluorescence confocal laser microscopy showed that the mutants had more intense fluorescence throughout the cell than the wild-type. Quantitative FCCS is a sensitive method to isolate and characterize carotenoid overproducing mutants and should be useful as a general method for the isolation of mutants increased in other fluorescent metabolites.


Unknown sequence amplification: application to in vitro genome walking in Chlamydia trachomatis L2.

A recently described technique, 'Chemical Genetics' unknown sequence amplification method, which requires only one specific oligonucleotide, has broadened the applicability of the polymerase chain reaction to DNA of unknown sequence. We have adapted this technique to the study of the genome of Chlamydia trachomatis, an obligate intracellular bacterium, and describe modifications that significantly improve the utility of this approach. These techniques allow for rapid genomic analysis entirely in vitro, using DNA of limited quantity or purity.


Manipulation of Corynebacterium glutamicum by gene disruption and replacement.

We have developed a system for the genetic manipulation of the amino acid-producing Corynebacterium glutamicum. Gene disruption and replacement were achieved by introducing, via conjugation, Escherichia coli vector plasmids carrying manipulated C. glutamicum DNA fragments. We obtained stable mutants in which the chromosomal lysA gene, encoding meso-diaminopimelate decarboxylase, was interrupted by a chloramphenicol resistance cartridge, or in which an essential internal part of the lysA gene was deleted. The deletion mutants retain neither antibiotic resistance markers nor vector sequences. This strategy is generally applicable to the construction of industrial strains to be used in fermentation processes.


Antibody engineering: advances from the use of Escherichia coli expression systems.

Synthesis in Escherichia coli of correctly folded antibody fragments that bind antigen with the same affinity as the whole antibody is now possible. Here I review the techniques for achieving this and the physical properties of the various fragments produced. This technology not only facilitates antibody engineering but is also the basis of screening libraries for binding activity. Although the immunization of animals has not been made unnecessary in the production of monoclonal antibodies, steps toward this goal are now feasible.


DNA amplification fingerprinting using very short arbitrary oligonucleotide primers.

The surprising finding that amplification of genomic DNA can be directed by only one oligonucleotide primer of arbitrary sequence to produce a characteristic spectrum of short DNA products of varying complexity, was applied as a strategy to detect genetic differences between organisms. This approach, DNA amplification fingerprinting (DAF), does not depend on cloning or DNA sequence information and can generate fingerprints from DNA of viral, bacterial, fungal, plant and animal origins. Primers as short as 5 nucleotides in length can produce complex banding patterns that are resolved by polyacrylamide gel electrophoresis and silver staining. Amplification fragment length polymorphisms (AFLPs) were detected between different human individuals as well as between soybean cultivars. It is anticipated that DAF will have wide application for DNA analysis.


Fermentation of a yeast producing A. niger glucose oxidase: scale-up, purification and characterization of the recombinant enzyme.

We have developed a fermentation process to produce up to 3 grams per liter of active, secreted glucose oxidase from a recombinant Saccharomyces cerevisiae. Real-time size-exclusion HPLC analysis is used to monitor enzyme production during fermentation, and purification to more than 95 percent is obtained using only filtration methods. The recombinant enzyme is stable to higher temperatures and a wider pH range than the native Aspergillus niger enzyme, and is free of contaminating amylase, cellulase and catalase.


A high containment polymodal pilot-plant fermenter--design concepts.

A 225 dm3 pilot-plant bioreactor system has been designed and constructed that is suitable for biohazardous fermentations. The design enables operation at containment levels above the requirements of good industrial large-scale practice (GILSP) without secondary containment of the whole plant. The main biosafety features of the systems include the use of steam barriers on O-ring seals, supply lines and stirrer seals, multiple O-ring seals, piping of condensate lines and pressure relief systems to a 'kill tank', double filtration of inlet and off gases and a mobile isolation unit that allows localised containment of sample valve and probe entry ports. The fermenter can, with minor modifications, be operated as a bottom-or top-stirred reactor for the culture of microbial or animal cells, or as an airlift reactor. The design offers considerable flexibility that could prove cost-effective for process development and production. The relevance of the various design features to enable bioreactor operations at pilot-plant scale to be carried out in compliance with current guidelines for large-scale culture of recombinant microorganisms and microbial pathogens is discussed.


Inhibition of trypsin by t-butyloxycarbonyl-alpha-aza-(4-aminophenyl)alanine phenyl ester.

t-Butyloxycarbonyl-alpha-aza-(4-aminophenyl)alanine phenyl ester (III: R = NH2) has been synthesized. The rate of inhibition of trypsin (EC 3.4.21.4) by this compound (due to acylation followed by slower deacylation) shows a marked pH maximum at approximately 6. The shape of the pH-rate curve is discussed in terms of (i) the normal pH-activity curve of trypsin reacting with a charged substrate, i.e. the protonated form of the amino compound, (ii) the deprotonation of the 4-amino group with pKa 4.3, and (iii) the lower rate of reaction of the enzyme with the uncharged, deprotonated form of the ester.


A yeast biosensor for glucose determination.

A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast Hansenula anomala is retained in calcium alginate gel on the surface of a glass electrode. This biosensor gives a Nernstian response in glucose concentration of 5 x 10(-4)-5 x 10(-3) mol/l with a response time of 5 min and a life-time of at least 2 months. Mannose and fructose are the only significantly interfering substances. The biosensor was used for measurement of glucose concentration in urine with results comparable to those obtained by a photometric enzymatic method.


Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor "Müller".

Streptomyces coelicolor "Müller" DSM3030 excretes a lysozyme comprising both beta-1,4-N-acetyl- and beta-1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozyme production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.


Dynamics of L-valine in relation to the production of cyclosporin A by Tolypocladium inflatum.

We report the kinetics of endogenous L-valine in the fungus Tolypocladium inflatum, in an effort to understand the enhancing effect of externally supplemented L-valine on the production of the immunosuppressant cyclosporin A (CyA) in chemically defined medium. In a batch laboratory stirred reactor cultivation, the concentration of intracellular L-valine increased by up to four times between the end of the exponential phase and the beginning of the stationary phase when the medium was supplemented externally with 4 g/l L-valine. The final CyA titre under these conditions was 710 mg/l compared to only 130 mg/l attained without L-valine supplementation. In contrast to substantial growth-associated production of CyA in unsupplemented culture, the formation of the immunosuppressant was prolonged during the stationary phase in L-valine-supplemented medium. As a result, the conversion yield of CyA on L-valine remained constant during the stationary phase at 0.27 g CyA/g L-valine.


Degradation of isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetic acid by a constructed Pseudomonas sp.

A 4-chlorobenzoate-degrading Pseudomonas sp. US1 was mated with a strain of Escherichia coli JMP 397 (harbouring the plasmid pJP4). An ex-conjugant designated Pseudomonas sp. US1 ex that could utilize all the isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetate was obtained. The ex-conjugant released stoichiometric amounts of chloride when grown on these chloroaromatics as sole sources of carbon and energy.


Fermentation and isolation of herbicolin A, a peptide antibiotic produced by Erwinia herbicola strain A 111.

Erwinia herbicola (Enterobacter agglomerans), belonging to the Enterobacteriaceae, produces the lipopeptide antibiotics herbicolin A and B, which are active against sterol-containing fungi. Fermentation of these antibiotics was performed in 20-l stirred glass fermentors in a batch process. Best yields of antibiotic production were found at low cultivation temperatures in a TRIS-buffered chemically defined medium. Under these conditions the amount of impurities aggravating the purification was minimized. Isolation was performed by adsorption, and gel and ion exchange chromatographic techniques. In a final purification step preparative high performance liquid chromatography (PHPLC) yielded pure herbicolin A.


Response of mammalian cells to shear stress.

The influence of shear forces on adherent mammalian cells was investigated by means of a developed flow chamber. The viability of the cells decreased with increasing exposure level and duration. Additional, changes in the morphology of the cells due to the shear forces were observed.


Automatic bioprocess control. 2. Implementations and practical experiences.

Our improved implementation for bioprocess control allows flexible responses to many process needs. It is based on computer equipment consisting of three hierarchically ordered levels. On the lowest level, a DDC slave computer handles setpoints and simple tasks generating the chemical and physical environment for the cells. It can be designed manually by the user or automatically by the supervisory computer on the second level. This provides for raw data organization, analysis and interpretation either to support personnel on line in decision making, to select predefined control strategies, or even to search for others. In the coordinating computer on the third level, common tasks of different supervisory computers (bioprocesses) are shared, saving money for the equipment. Tasks and concepts as well as experimental experiences are described to outline the capabilities of the configuration.


Dielectric spectroscopy as a novel and convenient tool for the study of the shear sensitivity of plant cells in suspension culture.

Plant cell suspensions of different species and different age were subjected to hydrodynamic stress while following the decline in the volume fraction of intact cells by measuring the permittivity of the cell suspension at radio frequencies. Results were compared with the fresh weight, dry weight, packed cell volume and cell number of the suspensions. At first a rapid decline is seen as the most shear-sensitive cells are broken up, followed by a slower decline as less sensitive cells are broken up. The sensitivity of the cells to shear stress depended strongly on the cell line used but only slightly on their age, older cells being more sensitive. The dependence of the shear sensitivity on the cell line might be an effect of the species investigated, the culturing conditions of the cell line, or both. It was found that cells that grow in a finely dispersed suspension are much less prone to shear stress than is often assumed.


Influence of low-temperature storage and glucose starvation on growth recovery in Escherichia coli relA and relA+ strains.

To study the influence of microgravity on bacterial growth behavior during a space mission, the special experimental conditions and the hardware environment necessitate storage of cells at low temperature, and permit a relatively short experimental period. Before this experimental period, cells have to recover their condition of steady-state growth, because it is only in this condition that the growth behavior of the flight and ground populations can be adequately compared. To meet these requirements and to obtain cells which recover rapidly their steady-state growth, we analyzed the size and shape of Escherichia coli cells during storage at 4 degrees C, with and without previous glucose starvation of the cells. It appeared that cells stored at low temperature in the presence of glucose continued to increase in average mass and assumed ovoid shapes. In addition, upon restoration of maximal growth rate at 37 degrees C, they continued to increase in size and showed a transient overshoot of their final steady-state value, which was reached after about 5 h. Cells previously starved for glucose, however, maintained their average size and rod-shape during low-temperature storage. Recovery of the starved cells was most rapid in the relA+ strain which, contrary to the isogenic relA strain, showed no overshoot and reached its final steady-state size within 2 h.


Genetic manipulation of Bacillus amyloliquefaciens.

Application of modern gene technology to strain improvement of the industrially important bacterium Bacillus amyloliquefaciens is reported. Several different plasmid constructions carrying the alpha-amylase gene (amyE) from B. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. The amyE gene cloned on a pUB110-derived high copy plasmid pKTH10 directed the highest yields both in rich laboratory medium and in crude industrial medium. The alpha-amylase activity, when compared with the parental strain, was enhanced up to 20-fold in the pKTH 10 transformant. This strain showed decreased activities for other exoenzymes, such as proteases and beta-glucanase suggesting common limiting resources in the processing of these enzymes. Deletions were made in vitro in genes encoding neutral (nprE), alkaline (aprE) protease and beta-glucanase (bglA). The engineered genes were cloned into the thermosensitive plasmid pE194, and the resulting plasmids were used to replace the corresponding wild type chromosomal genes in B. amyloliquefaciens by integration-excision at non-permissive temperature. The double mutant deficient in the major proteases (delta nprE delta aprE) showed about a 2-fold further enhancement in alpha-amylase production in the industrial medium compared with the relevant wild type background, [corrected] both when plasmid-free and when transformed with pKTH10; this strain also produced elevated levels of the chromosomally-encoded beta-glucanase; pKTH10 was stably maintained both in the wild type strain and in the delta nprE delta aprE mutant. We suggest that the higher yields in alpha-amylase and beta-glucanase in the delta nprE delta aprE strain are primarily due to improved access to limiting resources, and that decreased proteolytic degradation may have had a secondary role in retaining the high activity obtained.


Polyvinyl alcohol and polyethylene glycol as protectants against fluid-mechanical injury of freely-suspended animal cells (CRL 8018).

Two identical bioreactors run in parallel were used to examine the phenomenological characteristics of two additives, polyethylene glycol (PEG) and polyvinyl alcohol (PVA), used as protectants against fluid-mechanical cell damage. Cell-protecting ability was evaluated by comparing apparent cell growth rates of freely suspended CRL-8018 hybridoma cells cultured in serum-free medium under surface aerated conditions whereby cell damage is due to bubble entrainment and breakup. PEG of various molecular weights was used to determine whether the size of the polymer has significant effects on PEG's cell-protecting capabilities. All the PEG's with molecular weights larger than 1400 showed similar protective effects. The effect of PEG concentration was then evaluated and results showed that concentrations greater than 0.05% w/v did not significantly improve the cell-protecting properties. Direct comparisons made between the PVA, PEG, and pluronic F68 as cell protectants showed that PEG protected cells better than F68 and that PVA provided even better protection than PEG. The mechanism of protection, fluid-mechanical or biological in nature, was examined by growing the cells in additive from the beginning of the experiment (long-term exposure), or adding the additive after the cells had been agitated at rates detrimental to the cells (short-term exposure). In agreement with results reported previously on PEG and F68, fast-acting protection was seen. This implies a fluid-mechanical rather than a biological protection mechanism. In an attempt to correlate interfacial properties of the resulting media with shear protection, interfacial tension and viscosity measurements of all the media were made. On the basis of these measurements, we find no definitive correlations for evaluating these additives' cell-protecting capabilities.


Use of ars18 based vectors to increase protein production in Yarrowia lipolytica.

The isolation of ars sequence from the yeast Yarrowia lipolytica has recently been reported (Fournier et al., 1991). Vectors containing ars18 have been used to increase homologous and heterologous protein production. Examples presented are the Yarrowia lipolytica alkaline extracellular protease (AEP), the porcine alpha 1-interferon and the bovine prochymosin. A 2- to 6-fold increase in the corresponding protein production was observed and in several cases it was established that it corresponded to the copy number of plasmid in the cell.


The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei.

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.


Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.


Application of reversibly soluble polymers in bioprocessing.

Reversibly soluble polyelectrolytes, whose solubility in aqueous solutions is dependent on factors such as pH or ionic strength, may be used as supports for enzymes or affinity ligands. Their dual nature may be exploited to take advantage of the soluble form during, for example, enzymatic reactions, and the insoluble form during downstream processing operations for enzyme or product recovery.


Protein adsorption to solid surfaces.

The phenomenon of protein adsorption to solid surfaces affects the performance of many materials and processes, in areas ranging from medicine to biochemical engineering. Controlling protein adsorption, from solutions of single proteins as well as from more complex mixtures, requires an understanding of the mechanism(s) by which it occurs. This, in turn, entails detailed characterization of both the protein and the solid surface and identification of those factors controlling the adsorption process.


Conformational features of signal sequences and folding of secretory proteins in yeasts.

A yeast secretion system has been used extensively for the production of eukaryotic proteins that are expressed as non-native aggregates in E. coli. Secretory proteins translocate through the membrane using signal sequences, and fold in the cells (probably with the aid of several molecular chaperones). In combination with recent techniques of mutagenesis, this system has recently been implemented to study structure/function in signal sequences, and in vivo folding mechanisms of proteins. This review focuses on approaches to conformational features of signal sequences and folding of secretory proteins in yeasts.


New approaches to the study of tumor drug resistance.

The development of tumor drug resistance is the major obstacle to successful systemic chemotherapy. Therefore, devising methods for reversing drug resistance is a high priority and could lead to significant improvements in cancer treatment. The mechanisms of tumor drug resistance are manifold and are not well understood. The phenomenon of multidrug resistance (MDR) represents the development of resistance to most drugs, regardless of their chemical structure. Several types of MDR are known, for example, the overexpression of a cell membrane glycoprotein (P-170), increased activity of glutathione S-transferase, elevated levels of glutathione (GSH), and alterations in topoisomerase action. A partial reversal of tumor drug resistance has been achieved by the use of competitive inhibitors for the function of glycoprotein P-170, or by the inhibition of GSH synthesis; however, this strategy has not been substantially successful for improving the response of human tumors to clinical therapy. We have recently used electroporation, in conjunction with the cytotoxic drug, cisplatin (cDDP), in an attempt to circumvent drug resistance in cDDP-resistant mouse tumor cells (RIF/Ptr1). Electroporation is the application of a high-voltage electric shock which is known to create transient pores in plasma membranes of cultured cells. Electroporation plus cDDP treatment increased intracellular cDDP concentration and reversed cellular resistance to cDDP-induced cell killing.


Eukaryotic gene regulation: simple vs complex models.

The current generally accepted model of eukaryotic gene regulation is essentially a simple one. Regulatory proteins containing separable DNA binding and transcriptional activation domains, bind to specific DNA sequences in promotors and interact directly or indirectly with the TATA Box binding factor to increase the rate of transcription initiation at selected promotors. Here we present observations suggesting that the process may be more complex.


Targeting of gene expression to skeletal and cardiac muscle of trangenic animals.

The tissue restricted and developmental potentiation of transcription by chicken alpha-skeletal actin promoter regions fused to the reporter gene chloramphenicol acetyl transferase (CAT) were characterized in transgenic mice. Six of eight expressing transgenic mouse lines containing the chicken alpha-skeletal actin promoter fused to CAT resulted in preferential transgene transcription in skeletal muscle tissue, similar to the endogenous mouse alpha-skeletal actin gene. Two of the eight lines departed from the preferred pattern of skeletal muscle expression with primary expression of the transgene in the heart, a tissue containing primarily cardiac actin isoforms. Developmentally, a transition from embryonic heart to fetal and neonatal skeletal muscle expression was produced by the transgene promoter, a pattern of regulation similar to that of the endogenous alpha-skeletal actin gene. Instances of departure of transgene expression from the endogenous gene implied the existance of higher order muscle gene regulatory mechanisms.


Optimization of cell culture conditions for production of biologically active proteins.

We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 x 10(7) cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983). Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.


Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture.

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium. As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37 degrees C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640 + 10% FBS. It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable. Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast. Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2 x 10(8)/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day. The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.


Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas.

Two hybridoma systems, mouse.human-human (m.h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m.h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semi-continuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.


Establishment of vascular endothelial cell lines in a serum-free culture and the discovery of endothelin and a vasoactive intestinal contractor (VIC).

Immortal vascular endothelial cell lines were established and utilized for the production of an endothelium-derived contraction factor (EDCF) in a serum-free medium. After the discovery of Endothelin (21 amino acid peptide, ET) as an EDCF, a prepro ET cDNA isolated from human tissue was used to examine the expression of ET and its regulation in human endothelial cells. A gene family of ET was shown in mouse by using prepro ET cDNA as a probe. Thus, a novel peptide, Vasoactive Intestinal Contractor (VIC) homologous to ET was deduced from the sequence of one of these genes. VIC was confirmed to induce vasocontraction as well as intestinal contraction. Northern blot analysis indicated that this gene was expressed in the intestine but not in endothelial cells. A cloning and sequencing of prepro VIC cDNA from mouse intestine suggest that a VIC-like peptide, as well as VIC, are co-synthesized by cleavage from prepro VIC with 160 amino acids.


Serum-free cultivation of anchorage-dependent cells on microcarrier: effective production of human macrophage colony-stimulating factor.

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalizing the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10(7) cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.


Influence of system and molecular parameters upon fractionation of intracellular proteins from Saccharomyces by aqueous two-phase partition.

The interaction of molecular characteristics of proteins with the physicochemical properties of PEG-phosphate aqueous two-phase systems has been studied. This has involved characterization of protein molecular weight, charge, and hydrophobicity and study of PEG molecular weight and concentration, phosphate concentration, and pH. System characterization has been conducted in the context of limited stage fractionation procedures for protein recovery from baker's yeast. Results are presented which show that the degree of purification achieved is dependent on macromolecular surface properties rather than system operating conditions. A simple conceptual model of partitioning in PEG-phosphate aqueous two-phase systems is presented which is applicable in the rational design of fractionation procedures and serves to limit the amount of empirical experimentation necessary for the establishment of practical operations.


Immobilized enzyme system for determination of sialic acid in serum or urine.

An immobilized enzyme system has been developed and employed to determine the concentration of sialic acid (N-acetylneuraminic acid) in human serum and urine. Two enzyme pairs, neuramindiase-Neu-5-Ac lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized onto 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activity of the co-immobilized neuraminidase and Neu-5-Ac lyase were 60% and 78%, and those of pyruvate oxidase and peroxidase were 50% and 95% of the corresponding soluble enzymes, respectively. The optimal reaction pH at 37 degrees C for each of the co-immobilized enzymes was about one pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of each enzyme was increased as a result of immobilization. The thermal stability at 45 degrees C of the immobilized neuraminidase, Neu-5-Ac lyase, pyruvate oxidase, and peroxidase were increased 80-, 83-, 115-, and 147-fold, respectively. Km and Vm of each immobilized and co-immobilized enzyme have also been determined. The system provided a convenient and rapid method to determine the concentration of total sialic acid without pretreatment of the sample. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The co-immobilized enzymes were stable for at least 1 year of 500 tests when used repeatedly. The system is thus a reproducible and reliable novel assay method for sialic acid in the serum or urine sample.


Isolation and thermal stability studies of two novel serine proteinases from the fungus Tritirachium album Limber.

A number of serine proteinases are secreted into the culture medium when Tritirachium album Limber is supplied with protein as the only nitrogen source. From this population of proteinases, we have isolated two novel proteolytic enzymes, designated as proteinase R and T. We have compared the thermal stability of these two proteinases with that of subtilisin BPN' and proteinase K. Both of these proteinases were thermally stable in the absence of detergents in buffers of low (4.0) and high (10.0) pH. The thermal stability of proteinase T was not affected by the presence of 1.0% SDS either at pH 8.0 or 10.0 in contrast to proteinase R which became heat labile. At low pH, the presence of SDS was detrimental to the stability of all the proteinases.


Expression of active recombinant human alpha 1-antitrypsin in transgenic rabbits.

A DNA construct containing the human alpha 1-antitrypsin gene including 1.5 and 4 kb of 5' and 3' flanking sequences, was microinjected into the pronucleus of rabbit embryos. The recombinant human protein was (a) expressed in the blood circulation of F0 and F1 transgenic rabbits at an average concentration of 1 mg ml-1, (b) shown to be fully active and (c) shown to be separable from its rabbit counterpart. Transgenic rabbits might represent a novel source of human proteins of therapeutic interest.


Flavone glycosides from Asplenium normale.

Eight flavone glycosides were isolated from fronds of Asplenium normale, its two varieties, var. boreale and var. shimurae, and related species, A. oligophlebium. Six of the glycosides were identified: apigenin 7-O-dirhamnoside and 7-O-glucosylrhamnoside, luteolin 7-O-dirhamnoside and 7-O-glucosylrhamnoside, genkwanin 4'-O-glucosylrhamnoside, and vicenin-2. The remaining two glycosides were tentatively characterized as genkwanin 4'-O-glycoside and 6,8-di-C-glycosylluteolin. The four taxa analysed in this survey all had distinctive flavonoid profiles.


Scammonins I and II, the resin glycosides of radix scammoniae from Convolvulus scammonia.

The ether-soluble resin glycoside ('jalapin') fraction obtained from scammony roots, on alkaline hydrolysis, gave a glycosidic acid, scammonic acid A, together with isobutyric, 2S-methylbutyric and tiglic acids. In addition, two kinds of resin glycosides, named scammonin I and II, were isolated and characterized, respectively, as (11S)-hydroxyhexadecanoic acid, 11-[( O-6-deoxy-4-O-(2(E)-methyl-1-oxo-2- butenyl)-beta-D-glucopyranosyl-(1----4)-O-6-deoxy-2-O-(2-methyl-1-oxobut yl)- alpha-L-mannopyranosyl-(1----2)-O-beta-D-glucopyranosyl-(1----2)-6-deoxy -beta- D-glucopyranosyl]oxy)-, intramol. 1,3"'-ester and (11S)-hydroxyhexadecanoic acid, 11-[( O-beta-D-glucopyranosyl-(1----4)-O-6-deoxy-2-O-(2-methyl-1-oxobutyl)- alpha-L-mannopyranosyl-(1----2)-O-beta-D-glucopyranosyl-(1----2)-6-deoxy -beta-D - glucopyranosyl]oxy)-, intramol. 1,3"'-ester.


Kaempferol glycosides from Hosta ventricosa.

Leaves of Hosta ventricosa yielded eight kaempferol glycosides, of which six: the 3-(2G-glucosylrutinoside)-7-glucoside, 3-sophoroside-7-glucoside, 3-rutinoside-7-glucoside, 3-(2G-glucosylrutinoside), 3-sophoroside, 3-rutinoside were fully characterized and two: the 3-xylosylrutinoside-7-glucoside and 3-xylosylrutinoside were tentatively identified. Investigations included 1H-1H COSY and 1H-1H 2D J NMR analysis.


Clematoside-S, a triterpenoid saponin from the roots of Clematis grata.

Clematoside-S, a new triterpenoid saponin from the roots of Clematis grata, has been identified by chemical and spectroscopic methods as hederagenin-3-O-beta-D-ribopyranosyl (1----3)-alpha-L-rhamnopyranosyl(1----2)-alpha-L-arabinopyranoside .


Two flavonol glycosides from Chenopodium quinoa.

Two new flavonol glycosides from the seeds of Chenopodium quinoa have been isolated. Their structures were established as kaempferol 3-apiofuranosyl(1"'----2")rhamnopyranosyl(1""----6")galactoside and kaempferol 3-apiofuranosyl(1"'----2")rhamnopyranosyl(1""----6")galactoside. The main flavonoid glycoside was kaempferol 3-(2,6-dirhamnopyranosyl)galactoside.


A cardenolide tetraglycoside from Oxystelma esculentum.

A new cardenolide tetraglycoside, oxyline, was isolated from the dried roots of Oxystelma esculentum. On the basis of chemical and spectroscopic evidence the structure of oxyline was established as 3-epi-uzarigenin-3-O-beta-D-cymaropyranosyl-(1----4)-O-beta- D-thevetopyranosyl-(1----4)-O-beta-D-cymaropyranosyl- (1----4)-O-beta-D-digitoxopyranoside.


The amino acid sequence of ferredoxin from Hordeum vulgare.

Ferredoxin from barley consists of a single polypeptide chain of 97 amino acids, four of which are cysteine. These residues, which bind the iron atoms of the active centre, are in identical positions to those of other ferredoxins. The primary structure shows considerable similarity with other plant ferredoxins.


An anticoagulant fucoidan from the brown seaweed Ecklonia kurome.

The structure of a alpha-L-fucose-rich, sulphated polysaccharide (C-I) with a potent anticoagulant activity, which was isolated from the brown seaweed Ecklonia kurome, has been studied. Methylation analysis showed that C-I consisted mainly of 3-linked and 3,4-disubstituted fucopyranosyl residues in addition to non-reducing terminal fucofuranosyl and fucopyranosyl residues, 2,3-di- and 2,3,4-tri-substituted fucopyranosyl residues and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-I gave neutral di-, tri-, tetra- and highly polymerized-oligosaccharide fractions. GC-MS and methylation analysis indicated that di- and trisaccharide fractions consisted mainly of Fuc-(1----3)-Fuc and Fuc-(1----3)-Fuc-(1----3)-Fuc, respectively, in addition to small amounts of Fuc-(1----4)-Fuc, Fuc-(1----4)-Gal and Fuc-(1----3)-[Fuc-(1----2)-]Fuc. When methylated C-I was subjected to methanolysis for desulphation followed by remethylation with deuterated methyl iodide, most of deuteriomethyl groups substituted to position 4 of 3-linked Fuc.


12-Keto steroidal glycosides from the caudex of Yucca gloriosa.

Eight new steroidal glycosides, tentatively named YS-VI, -VII, -VIII, -IX, -X, -XI, -XII and -XIII were isolated from the caudex of Yucca gloriosa along with P-1, YG-2 and YG-3 previously obtained from flowers. The structures of five of these compounds were elucidated as mexogenin 3-O-beta-D-glucopyranosyl-(1----2)-beta-D-galactopyranoside (YS-VI), gloriogenin 3-O-beta-D-glucopyranosyl-(1----2)-[beta- D-glucopyranosyl-(1----3)]-beta-D-glucopyranoside (YS-VII) and 3-O-beta-D-glucopyranosyl-(1----2)-[beta-D-glucopyranosyl)-(1----3)]- beta-D-galactopyranoside (YS-VIII), manogenin 3-O-beta-lycotetraoside (YS-IX) and 3-O-alpha-L-rhamnopyranosyl-beta-lycotetraoside (YS-X), respectively, on the basis of chemical and spectral evidence.


Isolation of steroidal glycoalkaloids from Solanum incanum by two countercurrent chromatographic methods.

Using a bioassay for inhibition of plant growth and a combination of two countercurrent chromatographies: rotation locular countercurrent chromatography and droplet countercurrent chromatography, two biologically active glycosidal alkaloids, solasonine and solamargine were isolated from fresh ripe fruit of Solanum incanum. The combination of these chromatographic techniques has established an efficient isolation of polar phytochemicals of steroidal glycoalkaloids.


Brandioside, a phenylpropanoid glycoside from Brandisia hancei.

A new phenylpropanoid glycoside, brandioside, was isolated from Brandisia hancei. Its structure, [beta-(3',4'-dihydroxylphenyl)-ethyl]-(2-O-acetyl)-(3,6-O-di-alpha -L-rhamnopyranosyl)-(4-O-caffeoyl)-beta-D-glucopyranoside, was established by chemical and spectroscopic methods.


An anthraquinone and three naphthopyrone derivatives from Cassia pudibunda.

Chemical examination of the methanolic extract of the roots of Cassia pudibunda led to isolation of the new rubrofusarin-6-O-beta-D-glucopyranoside, quinquangulin-6-O-beta- D-apiofuranosyl-(1----6)-O-beta-D-glucopyranoside, quinquangulin-6-O-beta-D-glucopyranoside and chrysophanol dimethyl ether. Moreover the known chrysophanol, physcion, cis-3,3',5,5'-tetrahydroxy-4-methoxystilbene, trans-3,3',5,5' -tetrahydroxy-4-methoxystilbene, and cassiaside B were identified. The antimicrobial activity of some of these compounds is also reported.


Continuous aqueous phase extraction of proteins: automated on-line monitoring of fumarase activity and protein concentration.

Automated assay techniques are described for on-line measurements of fumarase activity and total protein concentration, including in-line sample dilution and sample multiplexing during continuous aqueous phase extraction. Fumarase was determined by following the conversion of L-malate to fumurate at a wavelength of 250 nm, while the protein assay was based on the Biuret reaction. Actual assay times of 2 and 4 min were achieved for the fumarase and protein measurements, respectively, with an effective measurement cycle time of 2 min. Standard deviations of c. 3.2 and 2% of the measured values were calculated for the enzyme and protein values, respectively. The assay system was coupled to a computer to allow on-line data visualization and storage.


Studies on the production of lipase from recombinant Staphylococcus carnosus.

Production of lipase from recombinant Staphylococcus carnosus pLipPS1 was studied in standard stirred tank bioreactors. Only low lipase activity was obtained under conventional operating conditions, i.e., moderate to high stirring speeds and aeration rates for keeping the dissolved oxygen concentration at high levels. Additional targetted experiments indicated that the reason for the observed low lipase activity is lipase inactivation due to surface forces and shear stress at the gas/liquid interface. Therefore, a cultivation strategy is proposed that minimizes gas/liquid interfacial area and maximizes the driving concentration for O2 mass transfer by controlling the dissolved oxygen to low values by gentle stirring and low aeration rates. Thus, high lipase activities can be obtained even in larger scale standard stirred tank bioreactors.


Molecular cloning and characterization of the recA gene of Methylomonas clara and construction of recA deficient mutant.

The recA gene of the methylotrophic bacterium Methylomonas clara has been isolated from a genomic library by hybridization with the Escherichia coli recA gene. Its complete nucleotide sequence consists of 1029 bp encoding a polypeptide of 342 amino acids. Nucleotide sequence analysis of the M. clara recA gene revealed extensive homologies to recA genes from E. coli and Pseudomonas aeruginosa. Part of the physiological activity of the M. clara RecA protein has become evident in that E. coli recA mutant HB101 is complemented. The cloned recA gene has been modified in vitro by site-specific mutagenesis and by insertion of a kanamycin-resistance gene cassette into the recA coding sequence. M. clara recA mutants were obtained by replacement of the active recA gene by an in-vitro inactivated gene copy.


Secretion of active truncated CD4 into Escherichia coli periplasm.

A truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli. The signal sequence from the E. coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter. The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120. Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane. A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E. coli can be optimized by manipulating the host secretion apparatus.


Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg).

A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 0.12-0.18 h-1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fed-batch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product formation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-1 culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.


Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes.

Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences. After optimizing the transformation procedure, transformation efficiencies of 15-21 transformants/micrograms plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the beta-tubuline gene of a benomyl-resistant Neurospora crassa mutant, the co-transformation rate was determined. Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies. In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions. The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies. Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus. Fractionation of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus.


The prospects for therapy with tumour necrosis factors and their antagonists.

Tumour necrosis factors (TNFs) alpha and beta are capable of causing dramatic necrosis of solid tumours. TNF-alpha has undergone clinical trials as an anti-cancer drug. Finding a role for TNFs in the treatment of cancer will depend on suppressing severe side effects and targeting the drug. Conversely, due to the participation of TNFs in inflammatory responses, antagonists of TNF-alpha are being considered for the treatment of several conditions, especially septic shock.


Human and mouse monoclonal antibodies by repertoire cloning.

Antibody repertoires, the wide range of antibody molecules produced by animals, can now be established in bacteria by cloning and expression of antibody genes. Beginning with immunized animals, antigen can be used to select, from the repertoire, clones which secrete specific monoclonal antibody. In the future, immunization may become unnecessary. The method may provide a general route, which has so far eluded biotechnologists, to human monoclonal antibodies.


Effect of NGF-2 on the survival of chick sensory ganglion neurons in vitro.

The biological activity of NGF-2, the third member of the NGF family, was investigated. Conditioned medium of COS cells transfected with expression plasmid for human NGF-2 cDNA promoted the survival of sensory neurons from dorsal root ganglia (DRG), and nodose ganglia (NG). Supernatant of mock transfected COS cells was less effective for the survival of DRG neurons and ineffective for the survival of NG neurons. These results suggest that NGF-2 is a novel neurotrophic factor whose biological activity is distinct from NGF.


Properties of a cell line (MEG-01SSF) of human megakaryoblastic leukemia cells.

MEG-01SSF cells were prepared from MEG-01S by conditioning in a chemically defined, serum-free medium. The cells grew well in the medium and required transferrin for growth. Doubling time was about 20 hours. The cells retained GpIIb/IIIa as a marker of megakaryocytes and transferrin receptors on the cell surface. Some of the cells in the stationary phase of growth produced platelet-like particles, which bore characteristic microtubule rings.


New perspectives on catalytic antibodies.

This review will give examples of the structure-function relationships used in the design of catalytic antibodies, including not only ligand or hapten-based approaches but also recent developments in the generation of catalytic antibodies through protein engineering. Last, a thermodynamic analysis is presented, which confirms that some antibodies do indeed catalyze their transformations by stabilizing transition states.


The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique.

We have previously demonstrated that the expression of fully functional Fv and Fab fragments in E. coli is possible by the simultaneous secretion of both chains to the periplasm. To increase production levels and facilitate engineering and random mutagenesis, we improved our previous vectors by introducing a resident repressor gene and a filamentous phage origin. We also developed a new purification strategy based on immobilized metal ion chromatography, with which a single-chain Fv fragment can be purified to homogeneity in a single step. We investigated the most efficient tail constructions and found that only a minimal structural change of three additional C-terminal amino acids is necessary. This modification has no deleterious effect on in vivo transport and folding or antigen affinity.


Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase.

We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.


Predicting the solubility of recombinant proteins in Escherichia coli.

We have studied the cause of inclusion body formation in Escherichia coli grown at 37 degrees C using statistical analysis of the composition of 81 proteins that do and do not form inclusion bodies. Six composition derived parameters were used. In declining order of their correlation with inclusion body formation, the parameters are charge average, turn forming residue fraction, cysteine fraction, proline fraction, hydrophilicity, and total number of residues. The correlation with inclusion body formation is strong for the first two parameters but weak for the last four. This correlation can be used to predict the probability that a protein will form inclusion bodies using only the protein's amino acid composition as the basis for the prediction.


Piezoelectric cell growth sensor.

We have developed a reusable piezoelectric sensor that enables rapid characterization of cell viability and response to cell-affecting agents. This is accomplished via a novel polymer transduction principle that involves reaction of a pH-sensitive amphoteric polymer with metabolically generated acid. Subsequent adhesion of the protonated polymer to the transducer surface causes a decrease in the sensor resonant frequency corresponding to the cell metabolic rate. This disclosure provides the first example of a piezoelectric sensor capable of detecting metabolic responses of viable cells. The sensor provides real-time measurement of cell metabolism and division rates, and antibiotic sensitivity. This technology provides the basis for an advanced piezoelectric sensor that does not require immobilized biological receptors and can be miniaturized without compromising signal-to-noise factors.


High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene.

We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus. In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture. The purified protein was as effective as native fragment C in immunizing mice. In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia. The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts). The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette. Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome. These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations. Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event. We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia.


A novel synthetic method for hybridoma cell encapsulation.

We report here what we believe is the first example of the encapsulation of hybridoma cells within a synthetic polymer by a simple gelation with dissolved cations in water, and at room temperature. Two lines of hybridoma cells were encapsulated within calcium cross-linked polyphosphazene gel microbeads without affecting their viability or their capability to produce antibodies. Interaction of these gel beads with the positively-charged polyelectrolyte, poly(L-lysine), of 102-kD molecular weight, produced a semipermeable membrane that was capable of retaining the cell-secreted antibodies inside the beads. Cell density increased 3.5-fold within 13 days concomitant with a 6.4-fold increase in antibody production. These synthetic membranes have the potential to aid in protein recovery schemes.


Actinorhodin production by Streptomyces coelicolor and growth of Streptomyces lividans are improved by the expression of a bacterial hemoglobin.

Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which can be limiting in large-scale fermentations. In an attempt to improve oxygen utilization by the cells, we expressed a heterologous bacterial hemoglobin gene in Streptomyces coelicolor and Streptomyces lividans. Hemoglobin expression was demonstrated by immunoblot analysis and carbon monoxide binding activity. In batch fermentations run under reduced aeration, the expression of hemoglobin in S. coelicolor resulted in a ten-fold increase in specific yields of the aromatic polyketide, actinorhodin. Actinorhodin yields were also much less sensitive to aeration conditions in the hemoglobin-expressing strain. In addition, hemoglobin-expressing S. lividans cells grown under reduced aeration had higher final cell densities and exhibited greater oxygen consumption rates than non-expressing cells.


High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.


Biotin formation by recombinant strains of Escherichia coli: influence of the host physiology.

Strains of Escherichia coli were transformed with different plasmids bearing the gene clusters bioXWF and bioDAYB isolated from the Gram positive bacterium Bacillus sphaericus. These genes encode for the enzymes involved in the metabolic pathway which synthesizes biotin from the precursor pimelic acid. Transformed E. coli strains were grown in bioreactors to reach a biomass of 18 g l-1 cell dry weight in 1 litre batch culture with substrate feeding and approximately 50 g l-1 in 10 l fed batch culture. Improved yields of total vitamers and biotin formed in these processes were achieved after a comparative analysis of different culture conditions, medium compositions, host strains and expression systems. Production of 27 mg l-1 of biotin and 200 mg l-1 of vitamers was achieved in 1 litre batch culture. Using a 10-1 fed batch process, biotin and vitamer concentrations reached maximum values of 45 mg l-1 and 350 mg l-1, respectively.


Physico-chemical characterization of the main factors affecting the mode of action of liver alcohol dehydrogenase immobilized on nylon tubing.

Alcohol dehydrogenase (ADH) from horse liver (EC 1.1.1.1), cross-linked through the bifunctional reactive glutaraldehyde, onto nylon tubing was immobilized (35 micrograms cm-2 internal surface of nylon tubing). ADH inactivation kinetics of the immobilized enzyme are of first order (t1/2 = 84.3 h, k = 8.2 x 10(-3) h-1 at 5 degrees C; t1/2 = 2.6 h, k = 0.26 h-1 at 50 degrees C). The activity versus pH profile points to a smaller effect of pH on the activity of the enzyme, which is the case of ADH in solution, explicable on the basis of limitations to proton diffusion towards/from the support. A limiting effect to free external diffusion of the substrate (products) towards/from the support was observed; this effect seems to determine the effective kinetic behaviour of immobilized ADH.