Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level. I. Steady-state responses to different glutamine feed concentrations.

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.


Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells.

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which codes for beta-galactosidase (beta-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10(-7), and 10(-6) M methotrexate (MTX), and the beta-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplified dhfr gene content and foreign beta-gal gene expression in the cell populations. A biochemical assay for beta-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10(-7) M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10(-6) M MTX was 20% lower than that of recombinant CHO cells at 10(-7) M MTX. There was no effect of 10(-5) M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect of dhfr and foreign gene amplification and increased beta-galactosidase expression.


Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads.

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.


Design and performance of a trickle-bed bioreactor with immobilized hybridoma cells.

A trickle-bed system employing inert matrices of vermiculite or polyurethane foam packed in the downcomer section of a split-flow air-lift reactor has been developed for hybridoma culture to enhance antibody productivity. This quiescent condition favoured occlusion and allowed the cells to achieve densities twelve fold greater (12.8 x 10(6) cells/ml reactor for polyurethane foam) than in free cell suspension. The reactor was operated in a cyclic batch mode whereby defined volumes of medium were periodically withdrawn and replaced with equal volumes of fresh medium. The pH of the medium was used as the indicator of the feeding schedule. Glucose, lactate and ammonia concentrations reached a stationary value after 5 days. With vermiculite packing, a monoclonal antibody (MAb) concentration of 2.4 mg/l was achieved after 12 days. The MAb concentration declined then increased to a value of 1.8 mg/l. In the polyurethane foam average monoclonal antibody (MAb) concentrations reached a stationary value of 1.1 mg/l in the first 20 days and increased to a new stationary state value of 2.1 mg/l for the remainder of the production. MAb productivity in the trickle-bed reactor was 0.3 mg/l.d (polyurethane foam) and 0.18 mg/l.d (vermiculite) in comparison to 0.12 mg/l.d for free cell suspension. This trickle-bed system seems to be an attractive way of increasing MAb productivity in culture.


Maximisation of perfusion systems and process comparison with batch-type cultures. Maximisation of perfusion cultures.

A comparison of cell yields and monoclonal antibody productivity from the same hybridoma has been made in a wide range of cell bioreactors including both batch and continuous perfusion cultures. The most productive systems were based on porous microcarriers in fixed and fluidised beds which can be operated with a high degree of efficiency and reliability from the physico-chemical engineering point of view. Further improvements should be possible by improving the physiological environment in dense cell cultures, as indicated by the preliminary studies that are described. These include experimental data showing the relationship between monoclonal antibody production rates with glucose, glutamine, ammonia, and oxygen levels in microporous beads. The results strongly indicate that perfusion processes that are scaleable in both volume and cell density can significantly reduce production costs. Manufacturers of biologicals from animal cells now have a choice between the proven batch-type processes and reliable perfusion systems based on microporous beads.


Modified CelliGen-packed bed bioreactors for hybridoma cell cultures.

This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG1 monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 10(8) cells per cm3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1%) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.


The membrane dialysis bioreactor with integrated radial-flow fixed bed--a new approach for continuous cultivation of animal cells.

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l-1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran carriers with 3-5 mm diameter showed an advantage compared to those with 1-2 mm diameter. For the 3-5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s-1 and 0.75 mm s-1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.


Optimal medium use for continuous high density perfusion processes.

For maintenance of high cell density in continuous perfusion processes not only feeding with substrates but also removal of inhibitors and toxic waste products are of special interest. High perfusion rates cause large volumes of product containing medium which have to be processed in product isolation. In order to minimize these volumes concentrated feed solutions of optimized medium are used. On the other hand, such media may cause high concentrations of toxic or inhibitory metabolites which can negatively influence cell growth and product formation. Especially, if the spent medium (or special parts of it) is used again after product isolation, the removal or even better the control of inhibitor production is of highest importance. We have developed a continuous fermentation concept and system (continuous medium cycle bioreactor, cMCB) in which both limitation and inhibition effects can be generated to identify special substances as limiting or inhibitory components. With the results from those experiments it was possible to lower the total perfusion rate during serum-free perfusion cultures of hybridoma cells and to obtain an optimal substrate utilization. The advantages for decreasing the production costs (for media, special supplements and product isolation) are obvious. The other aim of this study was to identify secreted metabolic waste products as inhibitor or toxic metabolite.


Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.


Expression of human alpha 1 antitrypsin in transgenic sheep.

We have recently described the production of large amounts (< or = 65 grams per litre) of enzymatically active human alpha 1 antitrypsin in the milk of transgenic sheep (Wright et al., 1991). Here, we describe in more detail the expression of the human protein in the milk of these animals throughout the lactation period. Human alpha 1 antitrypsin is also found at much lower levels in the plasma of transgenic ewes before, during and after lactation. It is also detected in male plasma at very low levels. We have previously shown human alpha 1 antitrypsin purified from transgenic sheep milk to be indistinguishable from commercially available human plasma derived alpha 1 antitrypsin in terms of gross sugar content and in vitro activity. Here we extend this comparison to more detailed analyses of glycosylation state, amino-terminal sequence, pI value, and molecular weight determination by mass spectrometry.


Specific monoclonal antibody productivity and the cell cycle-comparisons of batch, continuous and perfusion cultures.

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G1 phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G1 phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.


The role of laminin in attachment, growth, and differentiation of cultured cells: a brief review.

Laminin, a major structural multidomain protein of basement membranes, has been shown to exert a variety of biological activities. Prominent among those are mediation of cell attachment as well as influences on cellular proliferation, differentiation and motility. Distinct domains of laminin have been identified which carry these activities. The active sites on laminin are recognized by cellular receptors, several of which belong to the integrin class of heterodimeric transmembrane proteins. These are in direct contact with intracellular proteins and mediate signals from the extracellular matrix to the cytoskeleton and possibly to other intracellular regulatory systems. The biological activities of laminin may be used to design optimal conditions for the expression of a differentiated phenotype of cells in culture.


Comparison of culture methods and an immunofluorescence assay for the detection of Legionella pneumophila in domestic hot water devices.

The objective of this study was to compare an indirect immunofluorescence assay with culture methods for the identification of Legionella pneumophila serogroups 1 to 6 in hot water samples taken from domestic environments. Hot water samples were obtained from the water heater, the shower heads, and the most frequently used faucet of 211 private houses. Concentrated water samples were inoculated on buffered charcoal yeast extract agar (BCYE) and on a semi-selective culture medium (GPV). Colonies with a morphology similar to that of Legionellaceae were subcultured on BCYE and on blood agar plates; those that grew on the former but not the latter were further characterized and identified by direct immunofluorescence techniques. The concentrated samples were also smeared on multiple-well microscope slides and tested by indirect immunofluorescence with monoclonal antibodies against L. pneumophila, serogroups 1 to 6. Of the houses studied, 30% were found to contain culturable L. pneumophila in at least one water sample, whereas 63% were positive by indirect immunofluorescence. The sensitivity of this assay compared with culture varied from 16.7-21.1%, and its specificity was between 76.7% and 88.3% depending on the sample source (water heater, shower heads, or faucet). In the 38 houses with at least one positive sample found by both immunofluorescence and culture, total or partial agreement between serogroups identified by both techniques was only 34%. The results obtained in this study strongly suggest that indirect immunofluorescence is not an adequate alternative for the identification of L. pneumophila in hot water systems.


High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors in Pseudomonas stutzeri.

A number of Escherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained in Pseudomonas stutzeri ATCC 17588. A restrictionless mutant of P. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With the E. coli cloning vector pHSG298, transformation frequencies of up to 2 x 10(7) transformants/micrograms DNA were achieved. This frequency is comparable to that obtained for CaCl2-mediated transformation of E. coli; thus, direct cloning of DNA into P. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G + C genera in cases in which E. coli is not a suitable heterologous cloning host.


Characterization of Zymomonas mobilis alkaline phosphatase activity in Escherichia coli.

Zymomonas mobilis phoA gene encoding alkaline phosphatase was expressed in Escherichia coli CC118 carrying the recombinant plasmid pZAP1. The pH optimum for this enzyme was 9.0 and showed a peak activity at 42 degrees C. This enzyme required Zn2+ for its catalytic activity; however, Mg2+ or Ca2+ significantly affected the activity. This enzyme was found to be ethanolabile, and ethanol inhibition was reversed by addition of Zn2+. Kinetics of Z. mobilis alkaline phosphatase production in E. coli CC118 (pZAP1) showed that the enzyme activity was growth associated and localized in the cellular fraction, and the maximum activity was found in the stationary phase.


Phase variations in Bifidobacterium animalis.

Strains isolated from rabbit, chicken, and rat feces and from sewage and fermented milk products, all identified as Bifidobacterium animalis, were found to show phase variations in colony appearance and in cellular morphology. The rate of transition in a switching system from opaque to transparent colonies and vice versa was determined. Differences in protein components and in penicillin-binding proteins (PBPs) of the cells from different colony types are shown.


Change of speciation of Cu(II) in growth medium due to uptake of ammonia by Pseudomonas testosteroni during growth.

Growth of Pseudomonas testosteroni in a medium containing 1 mM Cu(II) causes a color change from blue to green. The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1 mM [Cu(II)] in the medium. The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and a d-d absorption at 720 nm. The absorption at 660 nm for the blue supernatant solution is probably due to [Cu(NH3)3(H2O)3]2+. Growth of the organism causes loss of ammonia and a speciation change to [CU(NH3)2(H2O)4]2+, with a shift in absorption maximum from 660 to 720 nm. These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth. Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously.


Anoxygenic degradation of aromatic substances by Rhodopseudomonas palustris.

Three strains of the phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris were isolated from different environments and were evaluated for their aromatic degradative potential under phototrophic conditions. All three strains (PFR1, PNR4, and MRL1) utilized benzoate, 4-hydroxybenzoate, 4-aminobenzoate, 4-aminophenol, cinnamate, ferulate, phloroglucinol, and 4-dimethylaminobenzaldehyde in the absence of exogenous CO2. 4-Aminobenzoate and 4-aminophenol served as a carbon and nitrogen source for all the three strains. Utilization of 4-aminophenol was enhanced in the presence of 4-hydroxybenzoate. Salicylate was utilized by PFR1 and MRL1 strains, and phenol was utilized by the MRL1 strain only in the presence of exogenous CO2.


Sensitization of oral bacteria to killing by low-power laser radiation.

Twenty-seven compounds were screened for their ability to sensitize Streptococcus sanguis to killing by light from a 7.3-mW Helium/Neon (HeNe) laser. Bacteria were mixed with various concentrations of the test compounds, spread over the surfaces of agar plates, and then exposed to light from the HeNe laser for various time periods. The plates were then incubated and examined for zones of inhibition. Those compounds found to be effective photosensitizers were then tested against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum. Toluidine blue O, azure B chloride, and methylene blue at concentrations of 0.005% (wt/vol) were effective photosensitizers of all four species, enabling killing of bacteria following exposure to laser light for only 30 s.


Correlation between the virulence of Francisella tularensis in experimental mice and its acriflavine reaction.

Correlation between the virulence of Francisella tularensis in experimental mice and its acriflavine reaction was studied. The cultures derived from all four strains (Ebina, CMB2, Schu, and N9) that had long been subcultured on agar media yielded two types of colonies, i.e., acriflavine reaction-positive (acf+) and acriflavine reaction-negative (acf-) colonies. All acf+ colonies, regardless of their parent strains, were shown to be low virulent in mice. Acf- colonies were shown to be either high (Ebina, CMB2) or low (Schu, N9) virulent. The low-virulent acf- colonies gained virulence during several passages in mice, whereas the acf+ colonies remained low virulent even after the animal passages.


A 28-kilodalton fibronectin-binding protein of group A streptococci.

Lipoteichoic acid (LTA) has been implicated as a major adhesin of group A streptococci that interacts with fibronectin (Fn). It has been suggested that protein adhesins may also be involved in the binding of Fn to streptococci. We searched for such a protein by transblotting membrane preparations from M types 5, 19, and 24 group A streptococci to nitrocellulose and reacting the blot with 125I-Fn. The Fn reacted with a 28-kDa polypeptide from all three serotypes of streptococci. Using affinity-purified antibodies to the 28-kDa protein in immunoblots of membrane preparations from various streptococci, we demonstrated that the 28-kDa protein is present in all 17 strains tested. Affinity-purified antibodies to the 28-kDa protein also reacted in varying degrees with intact streptococci, demonstrating that the antigen is exposed on the surface of intact organisms. Our results suggest that, in addition to LTA, group A streptococci contain a common Fn-binding moiety that is expressed as a major component of membrane preparations and that is accessible on the surface of streptococci for interactions with Fn.


Role of nalidixic acid in isolation of Salmonella typhimurium strains capable of growth at 48 degrees C.

Salmonella typhimurium thermotolerant mutants dependent on the presence of nalidixic acid for growth at 48 degrees C were isolated and designated nalidixic acid-dependent, thermotolerant mutants, naldttl. Genetic mapping revealed that naldttl alleles map within the gyrA gene. When S. typhimurium strain Q was plated in the dark on nutrient agar containing nalidixic acid (20 micrograms/ml) as a photosensitizer and briefly exposed to white light or near VU light prior to incubation at 42 degrees C, nalidixic acid-resistant mutants arose in about 16 h at frequencies of 5 x 10(-8) for white light and 1 x 10(-6) for near UV light. About 10% of these nalidixic acid-resistant mutants derived from photodynamic mutagenesis exhibited the thermotolerant characteristic.


Molecular cloning and expression of Bacillus subtilis bglS gene in Saccharomyces cerevisiae.

A 2.7-kb EcoRI DNA fragment carrying a Bacillus subtilis endo-beta-1,3-1, 4-glucanase gene (bglS) from the E. coli plasmid pFG1 was cloned into an Escherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transform Saccharomyces cerevisiae, and the bglS gene was expressed. Variation between levels of bglS gene expression in S. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found in B. subtilis, but the expression level of bglS gene in S. cerevisiae (YCSH) was much lower than that in E. coli (YCSH).


Purification and characterization of an aminopeptidase from Streptococcus mitis ATCC 903.

An aminopeptidase isolated from the cytoplasmic fraction of a cell extract of Streptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-beta-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-beta-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0-7.2 and at 37-40 degrees C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co2+ ions. Hg2+ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co2+ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co2+ ions.


Sequence analysis of an aphid endosymbiont DNA fragment containing rpoB (beta-subunit of RNA polymerase) and portions of rplL and rpoC.

The aphid Schizaphis graminum is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola). The nucleotide (nt) sequence of a 5040 base pair (bp) DNA fragment of B. aphidicola, homologous to the rplL-rpoB-rpoC portion of the Escherichia coli beta operon, was determined. The DNA coded for the terminal 35 amino acids of RplL (large ribosomal subunit protein L7/L12), the complete RpoB (beta-subunit of RNA polymerase), and the first 209 amino acids of RpoC (beta'-subunit of RNA polymerase). The deduced sequences of B. aphidicola RplL, RpoB, and RpoC were 71, 84, and 91% identical, respectively, to the homologous proteins of E. coli. The sequences of two portions of the intergenic region between rplL and rpoB were nearly identical in both B. aphidicola and E. coli. One sequence constituted an inverted repeat that could be an RNase III-messenger RNA processing site; the other sequence preceded RpoB. A compilation of the codon usage for RpoB, RpoC, and other B. aphidicola proteins indicated a major preference for A or T in the first and third positions, a result consistent with the low guanine plus cytosine (G + C) content of the DNA of this organism.


Cloning and expression in Escherichia coli of an alkaline phosphatase (phoA) gene from Zymomonas mobilis.

An alkaline phosphatase (phoA) gene from Zymomonas mobilis was isolated in Escherichia coli CC118 by use of the plasmid Bluescript KS+. The origin of the 6.4-kb DNA fragment in pZAP1 from the chromosome of Z. mobilis was confirmed by Southern blotting and hybridization studies. The Z. mobilis phoA gene was localized at one end of the chromosomal insert on plasmid pZAP1. The Z. mobilis phoA gene was expressed from its own promoter in E. coli, and the enzyme was localized to the periplasmic space. Z. mobilis alkaline phosphatase activity in E. coli was repressed in high-phosphate media and derepressed under a phosphate-limited growth condition. These results suggest that Z. mobilis alkaline phosphatase is subjected to normal regulation in E. coli.


Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli.

Whereas cell concentrations of 5-10 grams dry cell weight per liter (gDCW/l) are typical of batch cultures, fed-batch techniques can be used to achieve concentrations greater than 50 gDCW/l. Feeding strategies for fed-batch cultures include feed-back control as well as pre-determined feeding profiles. The volumetric yield of recombinant products can be improved by controlling the specific growth rate and the substrate concentration. Furthermore, inhibitory by-product formation can be minimized in fed-batch cultures. This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli. The modes of nutrient feeding that have been employed are discussed, and the factors important in attaining high cell concentrations as well as high specific yields of recombinant product are described.


An algorithmically optimized combinatorial library screened by digital imaging spectroscopy.

Combinatorial cassettes based on a phylogenetic "target set" were used to simultaneously mutagenize seven amino acid residues on one face of a transmembrane alpha helix comprising a bacteriochlorophyll binding site in the light harvesting II antenna of Rhodobacter capsulatus. This pigmented protein provides a model system for developing complex mutagenesis schemes, because simple absorption spectroscopy can be used to assay protein expression, structure, and function. Colony screening by Digital Imaging Spectroscopy showed that 6% of the optimized library bound bacteriochlorophyll in two distinct spectroscopic classes. This is approximately 200 times the throughput (ca. 0.03%) of conventional combinatorial cassette mutagenesis using [NN(G/C)]. "Doping" algorithms evaluated in this model system are generally applicable and should enable simultaneous mutagenesis at more positions in a protein than currently possible, or alternatively, decrease the screening size of combinatorial libraries.


Hyperthermostable variants of a highly thermostable alpha-amylase.

Genetic screening at temperatures between 70-80 degrees C far exceeds the range of growth of most bacteria, and is not applicable to isolate easily thermostable protein variants. We describe a temperature shift protocol and an in vivo screening method which allowed us to identify a hyperthermostable variant of the thermostable alpha-amylase from Bacillus licheniformis. Our strategy was to select, after hydroxylamine mutagenesis, an intragenic suppressor mutation which overcomes a mutation leading to a thermolabile enzyme. Sequence analysis of the mutated gene revealed only one change in the amino acid sequence, substituting a valine for alanine at position 209. This single amino acid replacement increased the half-life of the protein at 90 degrees C by a factor of two to three relative to the wild-type enzyme. When this substitution was combined with another stabilizing substitution (H133Y) we described previously, the stabilizing effects were additive. The half-life of the new protein was about 12 hours at 90 degrees C, corresponding to a nine to ten-fold increase over the wild-type enzyme and the industrial Bacillus licheniformis alpha-amylase Termamyl. These mutations are located in a predicted folding domain of the protein which appears crucial in determining thermal stability.


Importance of dietary cholesterol for the maturation of mouse brain myelin.

The effect of nil (control), 1% (CH-1) and 5% (CH-5) dietary cholesterol on the myelination of mouse brain, and its deposition in the heart and liver were investigated during infancy. Swiss Webstar female mice were given formulated diets from early gestation, and their pups were weaned on the same diet as that of the individual mothers up to 60 days after birth. The test diets increased the liver weight and cholesterol content compared to the control even in suckling pups (20 days), but did not significantly influence the heart weight until 60 days. The cholesterol content of the heart was not increased by the CH-1 diet throughout the feeding period, but it did increase the mole ratio of major myelin lipids and hastened its maturation. Myelin cholesterol was 10% higher in 20-day-old suckling pups in the CH-5 group compared to the control. Data indicate that dietary cholesterol altered the brain myelination rate of weaning mice, and that the mother's dietary cholesterol influenced myelination of the suckling pups.


Construction of a toxic insulin molecule: selection and partial characterization of cells resistant to its killing effects.

We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD50 of 1.1 x 10(-8) M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [125I]-insulin with an ED50 of 1.1 x 10(-8) M, which is identical to the ED50 of insulin (1.1 x 10(-8) M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [125I]-IGF-1 with a DTaI concentration of 1.1 x 10(-8) M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0 +/- 2.0 fmoles/mg protein down to 3.6 +/- 0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2 +/- 0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [125I]-insulin and no apparent difference in [125I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.


Purification and characterization of immunoglobulin production stimulating factor-II beta derived from Namalwa cells.

Two immunoglobulin production stimulating factors (IPSF) have been found in human Burkitt's lymphoma Namalwa cells. One IPSF named IPSF-II alpha was purified and identified as glyceraldehyde-3-phosphate dehydrogenase as previously reported. We report here purification, identification and characterization of IPSF-II beta. IPSF-II beta was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction column chromatography, anion-exchange column chromatography and gel filtration. The IPSF-II beta was estimated as a 46 KD monomeric polypeptide by gel filtration and SDS-PAGE. Partial amino acid sequence of the 46 KD protein was analyzed for 26 amino acid residues. The sequence very closely coincided with enolase (EC 4.2.1.11) derived from various origins and, it was completely homologous with that of human enolase alpha-chain. Rabbit muscle enolase stimulated IgM production of hybridoma lines, and IPSF-II beta had the enzymic activity. These results suggested that IPSF-II beta was alpha-enolase or its isozyme. IPSF activities of IPSF-II beta was stable in alkaline conditions whereas the enzymic activity was rapidly lost in alkaline conditions. Though IPSF-II beta stimulated IgM production of both human-human and mouse-mouse hybridoma lines in serum-free condition, it partially suppressed IgE production of mouse-mouse hybridoma lines.


Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures.

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be the result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.


Stable production of an analog of human tissue plasminogen activator from cultured Drosophila cells.

We have studied the expression of an analog of human tissue plasminogen activator, FK2P, in Drosophila Schneider 2 cells. A number of promoters were tested, including the Drosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE), Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1 alpha promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 micrograms FK2P/10(6) cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 microgram FK2P/10(6) cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominantly as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.


The growth of Vero cells in suspension as cell-aggregates in serum-free media.

Vero cell lines, usually considered anchorage-dependent, could be grown as cell-aggregates in suspension culture with serum-free media. Several different combinations of base media gave growth results above 10(6) cells/ml (NCTC 135:SFRE 199-1; NCTC 135:Waymouth MB 752/1; NCTC 135:RPMI 1640). Insulin was not essential for growth and Bovine Serum Albumin could be diluted out of the media if linoleic acid was present. The size and density of the aggregates formed varied depending on the media used.


Death of serum-free mouse embryo cells caused by transforming growth factor beta 1 and effects of nutritional factors.

Transforming growth factor beta 1 (1 ng/ml) caused death of serum-free mouse embryo cells cultured in a medium consisting of a 1:1 mixture of Dulbecco's Modified Eagle's medium and Ham's F12 medium supplemented with fibronectin, insulin, transferrin, epidermal growth factor, and high density lipoprotein. Cell death occurred in the presence of polyunsaturated fatty acids including linoleic acid in the absence of selenium. The death could be reversed by adding alpha-tocopherol to the culture indicating a mechanism involving fatty acid peroxidation. Butylated hydroxytoluene was a poor suppressor of cell death in contrast to alpha-tocopherol. High density lipoprotein and fatty acid-free albumin also suppressed cell death at the level of 20 micrograms/ml and 1 mg/ml, respectively. Transforming growth factor beta 1 also caused a low rate of cell growth after heat treatment of the cells at 45 degrees C.


Protein purification by counteracting chromatographic electrophoresis--on the disparity of focusing conditions.

The inconsistency in the focusing data on ferritin by the technique of Counteracting Chromatographic Electrophoresis, reported as flow rate vs potential gradient has been examined. Voltages in the gel region have been measured and analyzed, which are indicative of the fact that the potential gradients in the including and excluding gel regions vary from each other; those gradients are also different from the potential gradients in the buffer and polyacrylamide plug regions. Hence the existence of a constant voltage gradient (across the entire column) implied in the literature so far is not quite correct. The ionic strength of the carrier buffer, the chemical nature of the buffer components and the temperature of experimentation tend to obscure the 'true' data on stable accumulation zone formation.


Separation and purification of IgG1 and IgG2 from IgG-Cohn-II fraction of human plasma by pseudobioaffinity chromatography on histidyl-AH-sepharose.

L-histidine coupled to aminohexyl-sepharose (H-AH) has been used as an affinity sorbent to separate IgG from human plasma. Two subclasses IgG1 and IgG2 were specifically bound to histidyl-AH-sepharose at pH 7.4 and eluted using 0.2 M and 1M NaCl. The specificity of the two subclasses were determined by immunoelectrophoresis. Quantitative determination of IgG1, IgG2 was carried out using radial immunodiffusion technique.


Amplified expression and large-scale purification of protein G'.

PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.


A free-radical hypothesis for the instability and evolution of genotype and phenotype in vitro.

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., an in vitro evolution, which includes crisis, spontaneous transformation ('immortalization'), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by the in vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause of in vitro evolution. We propose an oxyradical hypothesis to explain each of the stages of in vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.


Techniques for the estimation of cell concentration in the presence of suspended solids.

Measuring cell concentration is of fundamental importance in many biochemical processes. However, this measurement is very difficult to make when solid particles are present along with the cells. This review examines strategies that have been used to estimate cell concentration in the presence of solid particles.


Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.

Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media.(ABSTRACT TRUNCATED AT 250 WORDS)


Fluctuations in continuous mammalian cell bioreactors with retention.

Continuous flow bioreactors with cell retention have been increasingly used for the cultivation of mammalian cells. The potential advantages of such bioreactors are high cell concentrations and volumetric productivities. In many reported cases, these systems have shown fluctuations in cell concentrations of various frequency and magnitude. To analyze the dynamics of the fluctuations, a model-based approach is followed. Simulations showed that large fluctuations in biomass resulted in response to fluctuations in the retention ratio when the system is operated at high dilution rate and high cell retention. The dependence of cell concentration fluctuations on variations in dilution rate and retention ratio was established by a cross-correlation statistical analysis on available experimental data. The slower dynamics and the fluctuation propensity of retention systems suggest that continuous culture without retention is more convenient for kinetic studies. In all likelihood, continuous culture with retention can be stabilized by controlling both the retention ratio and the dilution rate.


Mechanical properties of hydrocolloid gels filled with internally produced CO2 gas bubbles.

Agar (2%), alginate (1% algin), and kappa-carrageenan (1.5%) gel specimens were prepared from mother solutions that contained 0-2.5% sodium bicarbonate (agar and carrageenan) or calcium carbonate (alginate). Upon immersion in a citric acid bath (0-2%), the carbonate reacted with the diffusing acid to produce numerous carbon dioxide bubbles. The compressive strength and deformability of the gas-filled gels so produced were determined using a Universal testing machine and compared with those of pure gels and gels containing the carbonate but not subjected to the process after various immersion times. While the agar and alginate gels retained considerable mechanical integrity even after several hours, the carrageenan gels disintegrated after about 2-5 h. Under similar conditions, the number of bubbles produced in the agar gels was about twice that in the alginate gels, an observation that cannot be explained solely by stoichiometric considerations.


Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: evolution of neurohypophysial hormone precursors.

We have cloned and determined the nucleotide sequences of cDNAs encoding precursors of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), from the hypothalamus of masu salmon, Oncorhynchus masou. The deduced amino acid sequences of masu salmon VT and IT precursors (proVT-I and proIT-I) are highly homologous to those of chum salmon proVT-I and proIT-I, respectively. The VT and IT precursors are composed of a signal peptide, hormone and neurophysin (NP), the middle portion of which is highly conserved among vertebrates. Both the NPs extend about 30 amino acids at the C-terminal. The extended C-terminals have a leucin-rich segment in the carboxyl-terminal, as copeptin of vasopressin precursor. Southern bot analysis showed the presence of two types of proVT genes (proVT-I and proVT-II) and proIT genes (proIT-I and proIT-II) in an individual masu salmon, as in a chum salmon. Southern blot analysis with proVT probes further suggested that at least two different types of proVT-I genes exist in a single masu salmon. Northern blot analysis indicated that proVT-I and proIT-I genes are expressed in the hypothalamus, whereas proVT-II and proIT-II genes are not expressed. Evolutionary distance between proVT-I and proIT-I genes was statistically estimated based on synonymous nucleotide substitution in the coding region of the cDNAs. The magnitude of distance between masu salmon proVT-I and proIT-I genes suggested that the highly conserved central portion of NPs resulted from a gene conversion event. Between masu salmon and chum salmon, evolutionary distance for proVT-I genes is about 6-fold larger than that for proIT-I genes.


Affinity precipitation of proteins by surfactant-solubilized, ligand-modified phospholipids.

The use of ligand-modified phospholipids solubilized in aqueous solution by nonionic surfactant for affinity precipitation of proteins is described. Avidin was precipitated by contact with solutions in which dimyristoylphosphatidylethanolamine (DMPE) functionalized with biotin (DMPE-B) was solubilized in octaethylene glycol mono-n-dodecyl ether (C12E8) solutions. The nonionic surfactant solubilizes the phospholipid in micelles above its critical micelle concentration (CMC) and in small submicellar aggregates below this concentration. At C12E8 concentrations significantly exceeding its CMC, determined to be about 100 microM, precipitation of avidin by solubilized DMPE-B is not observed. In this regime, binding of protein by DMPE-B was monitored by a hyperchroic shift in the protein's UV-visible spectrum at 231.5 nm. The data were analyzed using a model that considers the four binding sites on the protein to be independent and identical in binding strength for DMPE-B. Below the CMC of C12E8, precipitation is observed and is monitored by increasing turbidity of the solution. The kinetics of precipitation and the aggregate size measured by quasielastic light scattering were analyzed using Smoluchowski kinetics and the Mie scattering theory. These results help establish more completely the factors that influence the precipitation of proteins by ligand-modified phospholipids, and they are helpful in specifying conditions for the precipitation of other proteins.


In situ studies of protein conformation in supercritical fluids: trypsin in carbon dioxide.

The conformation of the monomeric enzyme trypsin has been studied in supercritical carbon dioxide. Steady-state fluorescence spectroscopy is used to follow the conformation of trypsin in situ as a function of CO2 density. Our results show for the first time that protein denaturation can occur during the fluid compression step and that the native trypsin is only slightly more stable (1.2 kcal/mol) than the unfolded form. These results demonstrate the power of fluorescence spectroscopy as a tool for studying protein conformation and dynamics in supercritical fluids.


Occurrence of a novel 350-kDa serine proteinase in the fluid of porcine ovarian follicles and its increase during their maturation.

Porcine ovary was found to contain enzyme activities hydrolyzing peptide 4-methylcoumaryl-7-amide (MCA) substrates with a preference for Arg-MCA bond. The activities were shown to be present almost exclusively in the follicular fluid and to increase several times during follicular maturation. The enzyme responsible for these activities is thought to be a serine proteinase as judged from its strong inhibition by diisopropylfluorophosphate (DFP), leupeptin and antipain. The molecular weight of the native enzyme was electrophoretically estimated to be approximately 350,000, the result indicating that the enzyme is clearly distinct from plasmin (M(r) = 80,000) and collagenase (M(r) = 30,000-65,000), both of which are thought to be involved in ovulatory process. The substrate specificity of the partially purified enzyme was qualitatively different from that of plasmin. These results suggest that the enzyme is a novel type of serine proteinase.


Electrokinetic isolation of vesicles and ribosomes derived from Serratia marcescens.

Ribosomes and vesicles derived from the bacterium Serratia marcescens were separated from each other and from solubles using density gradient electrophoresis. Transport relationships were used to determine the electrophoretic mobilities of the particles. The effects of convection, sedimentation and diffusion were found to be negligible. The electrophoretic mobility obtained for the ribosome peak is -7 x 10(-5) cm2/(V.s). Under appropriate conditions, two vesicles peaks were obtained, the first with a mobility of -4 x 10(-5) cm2/(V.s) and the second with -9 x 10(-6) cm2/(V.s). This information can be used to predict the resolution of the separands in large-scale electrophoretic separations.


Perfluorooctyl bromide dispersions in aqueous media for biomedical applications.

In studying perfluorooctyl bromide (PFOB) dispersions in aqueous media, we have used two types of surfactant: egg yolk phospholipids (EYP) and polyglycerol esters (PGE). Our interest in these dispersions arises from their potential biomedical applications as imaging solutions and oxygen-carrying solutions (i.e., blood substitutes). For EYP systems, we have identified the dispersion structure as consisting of (a) PFOB droplets (250-nm diameter) stabilized by a phospholipid monolayer adsorbed irreversibly at the o/w interface and (b) small empty phospholipid vesicles. With both surfactants commercial preparations yielded stable systems, while purified samples, being non-dispersible, could not be made to act as emulsifiers. In both cases, minor components in the commercial surfactant were found to be necessary for the formation of a stable dispersion, enabling the transport of the pure surfactant to the PFOB/water interface.


Distribution of APGWa-immunoreactive substances in the central nervous system and reproductive apparatus of Helix aspersa.

The distribution of substances related to the tetrapeptide APGWa was investigated in the central nervous system (CNS) and the reproductive apparatus of Helix aspersa by immunocytochemistry. In the CNS, APGWa immunoreactive neurons were detected in all ganglia except the pedal ganglia. Concerning the mesocerebrum of the cerebral ganglia, only neurons of the right mesocerebral lobe reacted positively to the antiserum. In the genital apparatus, positive neurons fibres were seen in the muscular layer of the penis and, in the gonad, an immunoreactive material occurred on the heads of some spermatozoa. On the basis of these observations and of previous electrophysiological studies, an implication of AGPWa-like peptides in the control of mating behaviour is proposed. The significance of the positive reaction of the spermatozoa remains unclear.


Mass transfer in an airlift reactor with a net draft tube.

Gas-liquid mass transfer in an airlift reactor with net draft tube is investigated. The effects of both the ratio of draft tube to reactor diameter and the reactor pressure on oxygen transfer are considered. The value of the volumetric mass transfer coefficient, kLa, increases with a decreasing diameter ratio at higher air flow rates. The correlation of volumetric mass transfer coefficient with respect to the true superficial air velocity under different reactor pressures is determined. The kLa value decreases with increasing reactor pressure.


The solution of hollow fiber bioreactor design equations.

A methodology for simplifying the solution procedure for hollow fiber bioreactor design equations has been described. Such a procedure facilitates decoupling of membrane and spongy matrix equations from the tube side equations. The equivalence between the reduced equations and the hemodialyzer problem has been explicitly obtained.


Molecular methods for environmental monitoring and containment of genetically engineered microorganisms.

Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems.


The cellulose paradox: pollutant par excellence and/or a reclaimable natural resource?

The various aspects of cellulose as a pollutant are considered in view of its lack of toxicity on the one hand and its recalcitrant durable nature on the other. The microbial degradation of cellulosics is discussed, and the contrast between its success in handling natural cellulosic wastes versus its failure to cope with man-made refuse is described. Research carried out in the past decade has demonstrated that cellulolytic organisms are provided with cell surface multifunctional multienzyme conglomerates, called cellulosomes, which are capable of solubilizing solid cellulosic substrates. The intriguing properties of such complexes include their cohesive nature, their many enzymatic components, and a characteristic glycosylated cellulose-binding, 'scaffolding' component. The latter appears to serve as a substrate-targeting carrier, which delivers the other (hydrolytic) components to the cellulose. Progress in establishing efficient model systems for in vitro solubilization of purified cellulose or natural cellulosic substrates has been achieved using purified cellulosome preparations, fortified with beta-glucosidase and pectinase. The latter enzymes were required in order to alleviate the phenomenon of product inhibition which reduces the efficiency of the free cellulosome. Such combined enzyme systems are proposed as examples of future tailor-made cellulolytic systems for the degradation of natural cellulosics.


Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: an in vitro system with application to the bioartificial liver. Rat hepatocytes cultured in cylindrical collagen gels.

A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.


The influence of extra-cellular matrix and stroma remodeling on the productivity of long-term human bone marrow cultures.

The stromal cell layer is believed to play an important role in long-term human bone marrow cultures (LTHBMCs). At present, neither the role that the stromal cell extra-cellular matrix (ECM) plays in influencing stroma behavior is well understood nor are the effects of stroma aging. Rapid medium exchanged LTHBMCs were established on surfaces precoated with human natural fibronectin and type 1 rat tail collagen. Although initial adhesion of hematopoietic cells was improved by the presence of both ECMs, the overall progenitor and nonadherent cell productivity was not improved nor did the stroma grow to confluency faster. Thus, the ECMs used did not significantly influence the cell productivity of LTHBMCs. To examine the influence of stromal cell layer aging, conditioned medium was obtained from the first two weeks of LTHBMCs that was subsequently concentrated and used as a medium supplement in a second set of slowly exchanged LTHBMCs. The presence of the concentrated conditioned medium (conCM) enhanced the production of nonadherent cells three-fold compared with control over an eight week culture period. Control cultures that were exposed to conCM after 4 weeks in culture significantly improved their cell productivity during the latter 4 weeks of culture compared with control. The productivity of cultures exposed to conCM for 4 weeks dropped significantly when unsupplemented medium was used for the latter 4 weeks of culture. Interestingly, phytohemagglutin-stimulated leukocyte-conditioned medium stimulated LTHMBCs in a similar fashion, as did conditioned medium from early LTHBMCs. Taken together, these results strongly suggest that the stromal cell layer does produce important factors for active hematopoiesis during its growth to confluence.


Inactivation of proteins and other biological macromolecules during chromatographic methods of bioseparation.

The denaturation of proteins and other biological macromolecules such as gentamycin, mRNAs, and long-chain fatty acids during their separation by different chromatographic techniques is analyzed. Non-conventional techniques such as centrifugal partition chromatography are also examined. Particular attention is paid to the denaturing mechanisms prevalent under processing conditions, and how denaturation may perhaps be alleviated under laboratory conditions or during scale-up. The available mechanistic studies shed physical insights into the conformational behavior of proteins on chromatographic columns. Mechanistic studies of other biological macromolecule separation on columns is rare. Numbers for both recovery and purity of the biological product are presented wherever available. Scale-up studies are rare, nevertheless, those that are presented together do provide significant and valuable information, and may be generalized to other systems with caution.


Tissue engineering science: consequences of cell traction force.

Blood and tissue cells mechanically interact with soft tissues and tissue-equivalent reconstituted collagen gels in a variety of situations relevant to biomedicine and biotechnology. A key phenomenon in these interactions is the exertion of traction force by cells on local collagen fibers which typically constitute the solid network of these tissues and gels and impart gross mechanical integrity. Two important consequences of cells exerting traction on such collagen networks are first, when the cells co-ordinate their traction, resulting in cell migration, and second, when their traction is sufficient to deform the network. Such cell-collagen network interactions are coupled in a number of ways. Network deformation, for example, can result in net alignment of collagen fibers, eliciting contact guidance, wherein cells move with bidirectional bias along an axis of fiber alignment, potentially leading to a nonuniform cell distribution. This may govern cell accumulation in wounds and be exploited to control cell infiltration of bioartificial tissues and organs. Another consequence of cell traction is the resultant stress and strain in the network which modulate cell protein and DNA synthesis and differentiation. We summarize, here, relevant mathematical theories which we have used to describe the inherent coupling of cell dynamics and tissue mechanics in cell-populated collagen gels via traction. The development of appropriate models based on these theories, in an effort to understand how events in wound healing govern the rate and extent of wound contraction, and to measure cell traction forces in vitro, are described. Relevant observations and speculation from cell biology and medicine that motivate or serve to critique the assumptions made in the theories and models are also summarized.


Enzyme purification by immobilized metal ion affinity partitioning--application to D-hydroxyisocaproate dehydrogenase.

Extraction and purification of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei has been studied by means of immobilized metal ion affinity partitioning (IMAP) in aqueous two-phase systems. The partition of the enzyme can be influenced strongly by inclusion of iminodiacetic acid as chelating ligand coupled to polyethylene glycol and loaded with Cu2+ ions into the phase system. This applies to polyethylene glycol/dextran as well as polyethylene glycol/salt phase systems. An increase in enzyme partition coefficient of up to about 1000-fold was observed. Based on the mathematic model presented recently by Suh and Arnold (1990) approximately 6.4 histidine residues were calculated to be involved in the enzyme-metal chelate complex. Direct extraction of the enzyme from both cell homogenate and cell debris supernatant proved unsatisfactory due to disturbances caused by the presence of cell debris and low molecular weight cell components. A combination with a preceding prepurification by a fractional precipitation with polyethylene glycol resulted in a strong affinity effect accompanied by an efficient purification during IMAP (purification factor of 11 with a yield of approximately 90%). Based on this step, an efficient downstream process can be designed for D-hydroxyisocaproate dehydrogenase.


A reversed-phase HPLC method for measurement of 5-hydroxymethyl furfuraldehyde and furfuraldehyde in processed juices.

An HPLC method using a reversed-phase macroreticular PLRP-S column and phosphate buffer as eluent is described for the analysis of L-ascorbic acid degradation products, 5-hydroxymethyl furfuraldehyde and furfuraldehyde, in processed fruit juices. Measurement of the levels of 5-HMF and furfuraldehyde in citrus juices against time showed the presence of 5-HMF (0.45 mg l-1) even at zero time. An assessment on the effect of the additives on the formation of 5-HMF of reconstituted single-strength orange juice showed virtually the same results for all the samples stored at 4 degrees C and 20 degrees C, irrespective of the additive. For citrus juice samples which had been subjected to accelerated degradation, those that showed the highest decomposition of L-ascorbic acid, produced the highest level of 5-HMF. The presence of furfuraldehyde in any of the samples was not detected, probably due to the fact that furfuraldehyde was formed in such small amounts which are below the minimum detectability limit of the method (0.050 mg l-1).


Stabilities of antigen and antibody under elution conditions in immunoaffinity chromatography using monoclonal antibody.

In immunoaffinity chromatography using monoclonal antibody, the elution conditions of an antigen, recombinant alpha-amylase, were studied. From among various conditions, three elution methods that gave fairly good yields of antigen activity (pH 2.3-2.5, pH 12.3-12.5 and 0.1 M lithium 3,5-diiodo salicylate [LIS]) were selected and the stabilities of the antigen and the antibody were analyzed. The antigen seemed to be eluted from the immunoadsorbent due to partial denaturation of either the antigen or the antibody. LIS seemed to be a specific denaturant for the antibody and its action was reversible. In terms of the stability of the antigen and repeated use of the immunoadsorbent, LIS seemed to be the best reagent for elution in immunoaffinity chromatography.


Principles of the design and operational considerations of large scale high performance liquid chromatography (HPLC) systems for proteins and peptides purification.

This review introduces concepts of design of large scale HPLC systems for purification of proteins and peptides. It is addressed to users of large scale HPLC systems to aid in system selection and help in customizing the design. Major techniques used for large scale HPLC purification of proteins and peptides are briefly reviewed. Engineering aspects of system design are discussed in detail. The review of selected relevant literature is provided as well as author's experience with the existing designs and his own systems. Commercial publications have been used in preparation of this review but only the most significant are listed as examples. The design process for any new system should be related to the performance of existing systems, if possible of a large scale. Laboratory data are also very useful in aiding the design process since they provide a lead, as to which chromatography techniques may succeed in providing required separation levels. The expertise needed for system design and operation comes from many areas: protein and peptide chemistry, chromatographic theory, mass transfer and hydrodynamics, machine design and material science. All these factors have to be blended together during the system design process. The controls must ensure flexibility in adapting to changing system configuration, depending on the operational requirements for various products. Extensive interfacing with the operator during the process run is essential. This work is focused mostly on system design and operation for reversed-phase chromatography and hydrophobic interaction chromatography, but it also covers aspects associated with other chromatographic techniques. The reviewed design principles would also apply to design other than HPLC large scale chromatography systems for biotechnology and pharmaceutical operations.


Separation of beta-casein peptides through UF inorganic membranes.

Ultrafiltration of peptide mixtures is studied under various operating conditions (transmembrane pressure, tangential flow-rate) using two ultrafiltration inorganic membranes M5 and M1 with molecular weight cut-offs, MWCO 10 and 70 kD, respectively. It is shown that the separation of peptides is controlled by a dual mechanism: size exclusion and electrostatic repulsion. When the ionic strength is high enough to screen out the electrostatic interactions, experimental data are in good agreement with a sieving model developed to estimate the intrinsic transmission from the molecular weight of a component and from the MWCO of the membranes. Although the transmission so found is altered by concentration polarisation and pore blocking mechanisms, the results explain the apparent low transmission of peptides by ultrafiltration membranes. If the ionic strength of the fluid is low, electrostatic interactions can influence the transport phenomena, provided that the molecules are highly charged (at pHs away from the pI). For attractive interactions, an apparent partition coefficient larger than 1 is observed. Otherwise, the transmission is lower than predicted by the sieving theoretical equation, as if the partition coefficient were smaller than 1.


Adsorption of Streptococcus faecalis on diatomite carriers for use in biotransformations.

Adsorption of cells on particulate carriers is potentially one of the most cost-effective immobilization techniques available. Diatomite carriers, such as Celite, have desirable physical properties, are inexpensive, and are suitable for both mycelial and bacterial systems. This work investigated the use of diatomite carriers as a biocatalyst support in a packed-bed reactor where L-tyrosine was enzymatically decarboxylated using adsorbed, non-growing cells of Streptococcus faecalis. Composition of microbial adsorption on different Celite types, with mean pore sizes ranging from 0.55 to 22 microns, showed there was no significant difference in biomass loading capacity under the conditions used. Using Celite 560, biomass loadings in a packed-bed reactor varied from 10 to 30 g dm-3 of reactor volume, which compares favourably with other adsorption methods. When used to decarboxylate L-tyrosine, the reactor was found to have a half-life of 15-20 h. A combination of enzyme activity loss and slow leakage of biomass from the packed-bed reactor was responsible for the decline in conversion. Treatment of the S. faecalis cells with glutaraldehyde significantly reduced the enzyme activity loss and extended the reactor half-life to 65 h, but had little effect on the rate of cell leakage from the reactor. Further work on reduction of cell leakage rate seems necessary for evaluation of the system's practicality.


Expression of virally-transduced mouse tyrosinase in cultured chick embryo cells.

A cDNA encoding mouse tyrosinase was inserted into a plasmid containing the provirus of a replication competent Avian Leukosis Virus (ALV). A viral stock produced from the plasmid was used to infect cultured tyrosinase-negative (ca/ca) unpigmented chick embryo pigment cells. Five days after infection many cells were producing very dark discrete pigment granules. Cultures of tyrosinase positive, sex linked albino (sal) pigment cells produced no additional pigmentation. White Leghorn pigment cells responded to viral infection like the sal pigment cells.


Flocculation: an alternative process to ion-exchange chromatography: (A scale-up study using recombinant human superoxide dismutase as model protein).

Flocculation using pellicular charged flocculants was investigated as an alternative process to conventional chromatography in purification of recombinant proteins using human recombinant superoxide dismutase expressed in E. coli as a model system. The removal of pyrogens, proteins, debris and the yield were determined. At laboratory scale, the starting conditions were optimized to yield a stable solution and the flocculation process fitted into a purification scheme. 100 L fermentation broth was the initial volume at pilot scale. The process parameters determined at the laboratory scale were tested and the results were compared to the pilot scale. The method was also compared to an ion-exchange chromatography.


Temperature sensitivity of equine herpesvirus isolates: a brief review.

This article reviews the findings on temperature sensitivity of equine herpesvirus isolates with an emphasis on equine herpesvirus 3, etiological agent of equine coital exanthema. The hypothesis is presented that the relative apathogenic nature of this herpesvirus may be an indirect result of its inability to synthesize and/or process glycoproteins needed by the virus to produce infectious virions at the normal body temperature of its natural host. It is suggested that equine herpesvirus 3 is the more evolved and naturally attenuated member of the equine herpesviruses.


Preparation of peptide mixture with high Fischer ratio from protein hydrolysate by adsorption on activated carbon.

A peptide mixture with a high Fischer ratio (a molar ratio of Val + Leu + Ile to Phe + Tyr) was prepared by the adsorptive separation of a casein hydrolysate by activated carbon. The effects of the pH and ethanol content of the hydrolysate on the Fischer ratio and on the yield of the resulting peptide mixture were examined. A peptide mixture with the Fischer ratio of 31.6 was obtained at pH 2.5 without the addition of ethanol. The Fischer ratio was close to the ratio of the infusion solution of free amino acids that is now used for patients with liver diseases.


A synthetic peptide representing the thrombin receptor-binding domain enhances wound closure in vivo.

Our studies of alpha-thrombin as a growth factor have led to the development of a synthetic peptide (p508) that in vitro competes with thrombin for binding to high affinity receptors, and enhances mitogenic activity. To determine if this peptide could be used to accelerate wound closure in vivo, full thickness 6 mm dermal biopsy wounds on the dorsal skin of anesthetized rats were treated with p508 peptide, thrombin or PBS as control. At day 7, the p508 treated wound areas were 20% to 50% smaller than either thrombin or PBS treated wound sites. This suggests that p508 enhances aspects of wound healing, and avoids the normal in vivo regulatory mechanisms of intact thrombin.


Recovery of extracellular human insulin-like growth factor-I and II as a fusion protein from Escherichia coli culture broth by aqueous two-phase extraction.

The primary purification of human insulin-like growth factor-I (IGF-I) and IGF-II, produced extracellularly in Escherichia coli as a fusion to two domains (ZZ) derived from staphylococcal protein A, has been studied. First, the partitioning of IgG-affinity purified ZZ-IGF-I and ZZ-IGF-II, respectively, to the top phase in poly(ethylene glycol)/potassium phosphate aqueous two-phase systems were investigated. Thereafter, the extraction of ZZ-IGF-I with a poly(ethylene glycol) 1500/potassium phosphate system was performed directly in the bioreactor after the cultivation. This resulted in a reduction of the cultivation volume more than 3-fold with a recovery of about 90% of target protein in a poly(ethylene glycol)-rich phase. The majority of the cells partitioned to the potassium phosphate-rich bottom phase, while a smaller fraction was collected at the interface, and/or as a densely packed cake on top of the interface. Contaminating proteins were also eliminated to some extent, which resulted in an almost 2-fold protein purification. Some obvious benefits offered by the aqueous two-phase system in the primary purification have been demonstrated: Firstly, the possibility to an early process volume reduction and thereby a concentration of the target protein. Secondly, a simultaneous protein purification was achieved. From this work it can be concluded that aqueous two-phase extraction should be considered as an attractive candidate for the primary steps during the design of new purification processes for extracellular proteins.


Specificity of nucleotide sequence in DNA cleavage induced by D-glucosamine and D-glucosamine-6-phosphate in the presence of Cu2+.

32P-End-labeled restriction fragments derived from pBR322 and pUC9 DNAs were reacted with D-glucosamine or D-glucosamine-6-phosphate in the presence of Cu2+, and, after being heated at 90 degrees C in aqueous piperidine, the DNA products were analyzed on polyacrylamide gels for the sequence-specificity of alkali-labile cleavaged sites. The intensity of oligonucleotide bands of cleavaged sites was directly proportional to the concentration of aminosugars, indicating that the DNA cleavage was caused by the action of aminosugars themselves. The preferred DNA cleavage sites induced by these aminosugars were identical, both at pyrimidine-purine (5'----3') sequences, especially at thymineguanine ones, and to some extent at pyrimidine-pyrimidine (5'----3') sequences. The 6-phosphate moiety of D-glucosamine did not affect the specificity of DNA cleavage.


Immobilization of enzymes to porous-bead polymers and silica gels activated by graft polymerization of 2,3-epoxypropyl methacrylate.

Three types of organic polymers and bead-shape silica gels were activated by graft polymerization of 2,3-epoxypropyl methacrylate; in some cases, epoxide groups on the support surface were modified to NH2 groups. Eight active matrices so obtained were assessed as supports for immobilized enzymes using peroxidase, glucoamylase and urease. The immobilization yield of protein and specific activities of enzymes were better with supports containing NH2 groups than with those containing epoxide spacer arms. Maximum enzyme immobilization and storage stabilities were obtained with silica-gel beads activated by graft polymerization of 2,3-epoxypropyl methacrylate. With all eight matrices tested, the immobilized enzymes showed good stability with not less than 82% of the original activity persisting after 28 days. The developed matrices have potential for use in process-scale biotechnological operations.


Yeast alcohol dehydrogenase bound to membranes: surface and microenvironment effects on activity and stability.

The enzyme, yeast alcohol dehydrogenase, was adsorbed to porous nitrocellulose and nylon membranes. The two membranes provide different surface chemistries as indicated by the results of the streaming potential, enzyme adsorption, and fluorescein isothiocyanate adsorption experiments. The stability of the enzyme, as determined by continually measuring the extent of coenzyme reduction as a function of time, appeared to be much less for the enzyme adsorbed to the positively charged membrane surface. Moreover, the enzyme adsorbed to the positively charged membrane was the least responsive to pulses of the reducing agent, dithiothreitol, and appeared to exhibit the highest transition temperature when subjected to differential scanning calorimetry analysis. These results indicate that the entropically spreading process observed for other adsorbed proteins may be occurring and the process is more rapid and extensive when enzyme is adsorbed to the nylon than the nitrocellulose membrane. In addition to the relative stability of the enzyme on two different surfaces being examined, the effect of the microenvironment on modulating the activity of the enzyme was investigated by using the reversibility of the enzyme-catalyzed reaction as a probe of the average local environment of the enzyme. It was found that a threshold buffer concentration existed that, once exceeded, the effect of proton production by the reaction could be suppressed.


Oligonucleotidase activity of phosphodiesterase from the fruit body of Flammulina velutipes.

A phosphodiesterase (EC 3.1.4.1) was purified to homogeneity from the fruit body of Flammulina velutipes. The enzyme had considerable activity toward oligonucleotides. The Km values were 0.66 mM for ApA, 2.47 mM for (Ap)2A, and 3.03 mM for (Ap)3A. The enzyme hydrolyzed oligodeoxyribonucleotides as well as oligoribonucleotides. The oligoribonucleotides bearing a phosphate residue at the 3' end were not hydrolyzed by the enzyme. The enzyme hydrolyzed the oligoribonucleotides exonucleolytically from the 3' to 5' end. Thus the PDase of F. velutipes is considered to function in vivo as an oligonucleotidase (EC 3.1.13.3), which efficiently converts oligonucleotides to 5'-mononucleotides in the cell.


Effect of inclusion body production on culture turbidity and cell dry weight in growing bacterial cultures.

An approach to the optimization of product yield in an inducible inclusion body-producing system is presented. Following induction by indoleacrylic acid (IAA) of a trpLE-HIVgp41 fusion protein, we found a large increase in culture turbidity and single-cell dry weight. After an initial transition phase, new and constant values for specific growth rate, single-cell light turbidity, and single cell dry weight were achieved, allowing for the determination of optimal conditions for product formation.


Genetic engineering approach to toxic waste management: case study for organophosphate waste treatment.

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled-up to the pilot scale. The cell-free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.


Solubility as a function of protein structure and solvent components.

This review deals with ways of stabilizing proteins against aggregation and with methods to determine, predict, and increase solubility. Solvent additives (osmolytes) that stabilize proteins are listed with a description of their effects on proteins and on the solvation properties of water. Special attention is given to areas where solubility limitations pose major problems, as in the preparation of highly concentrated solutions of recombinant proteins for structural determination with NMR and X-ray crystallography, refolding of inclusion body proteins, studies of membrane protein dynamics, and in the formulation of proteins for pharmaceutical use. Structural factors relating to solubility and possibilities for protein engineering are analyzed.


D-ribulokinase from Klebsiella pneumoniae for continuous production of D-(-)-ribulose-5-phosphate.

The production of D-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to D-ribulose was catalysed by D-ribulokinase isolated from Klebsiella pneumoniae. For production of D-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for D-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of D-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of D-ribulose-5-phosphate l -1 per day were reached.


Quinoproteins: a new class of enzymes with potential use as biosensors.

The field of the enzymology of quinoproteins is still in its infancy. As more progress is made toward understanding the mechanisms of catalysis and electron transfer by protein-bound PQQ, it will become possible to more logically design specific quinoprotein-based electrodes. The characterization of new quinoproteins is progressing at a rapid rate. As new quinoproteins are found, the potential range of applications for use of these enzymes in biosensors will increase as well.


Affinity chromatography supports: a look at performance requirements.

Because of its high selectivity, affinity chromatography is a preferred tool in the downstream processing of high-value proteins and peptides of therapeutic interest. This review examines the affinity supports currently available, and investigates the performance characteristics and properties required of the support matrices for improved affinity-based supports for large-scale purification of biomolecules. Parameters for optimizing an affinity chromatographic process, and the advantages of affinity-based separation for scaled-up systems are highlighted.


Production of 5'-ribonucleotides by enzymatic hydrolysis of RNA.

Study of optimal operational conditions for RNA enzymatic hydrolysis to obtain 5'-ribonucleotides has been carried out. RNA from brewer's yeasts, obtained by ammonium extraction, was hydrolysed by a partially purified 5'-phosphodiesterase from barley rootlets. Temperature of 60 degrees C and pH 7 have been determined as the best operational conditions. Low RNA initial concentration (approximately 0.1%) and reaction time (approximately 1 h) have been identified as necessary to obtain a good yield of 5'-ribonucleotides.


Transfection of mouse cells with thymidine kinase gene of herpes simplex virus.

A mouse cell line (LP1-1) was established from the murine L cells deficient in thymidine kinase (L-M(TK-] by prolonged selective culture on the hypoxanthine-aminopterine-thymidine (HAT) medium following transfection with the thymidine kinase gene of herpes simplex virus type-I (HSVTK). Southern blot analysis has shown that the viral TK gene was integrated into one of the chromosomal loci by a single copy. From this established cell line, the 5-bromo-2-deoxyuridine (BrdU) resistant revertant was brought out at a frequency of 1 x 10(-6) and from these BrdU resistant revertants (LP1BU), one out of 1 x 10(5) cells could return to the HAT-resistant phenotype. The established LP1-1 cell line showed a typical biphasic nature of DNA synthesis as determined by the 3H-thymidine incorporation test. The activity of thymidine kinase was shown to be equivalent to that of the DNA polymerase-alpha when the whole nuclear fraction or the nuclear matrix were used for examination. These results indicate that the transfected viral TK gene can be expressed under the normal cell-cycle regulation and its gene product can act as a component of the multienzyme complex which is responsible for DNA replication.


Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty.

Distribution of a delta (delta) sequence of the Ty element on a chromosome of the yeast Saccharomyces cerevisiae was analysed by pulsed-field gel electrophoresis. More than 100 copies of the delta sequence were nonrandomly distributed on the chromosome. Using the delta sequence as a recombination site, mouse alpha-amylase and human beta-endorphin genes were introduced into the chromosomal DNA. The integration occurred on a particular chromosome in each case and the copy number was estimated as three to five. It was also found that single- or multi-copy integration occurred at a single or multiple sites on the particular chromosome. The integrants secreted alpha-amylase and beta-endorphin by three-to fivefold compared with single-copy integrants. This type of integration was mitotically stable over a period of 50 generations under non-selective conditions.


A human-human hybridoma secreting anti-Pseudomonas aeruginosa exotoxin-A monoclonal antibody with highly potent neutralizing activity.

A hybridoma secreting human monoclonal antibody (MAB) against Pseudomonas aeruginosa exotoxin A (PEA) was constructed by fusing Epstein-Barr virus-transformed peripheral blood lymphocytes with human B lymphoblastoid cell line TAW-925. The human-human hybridoma stably produced human IgG2 MAB at the rate of 0.4-0.5 microgram/ml per 10(6) cells per day for more than six months, and the MAB was capable of neutralizing the in vitro cytotoxic and in vivo lethal effects of PEA with approximately 100- and 70-fold, respectively, higher activity than serum polyclonal antibody preparations.


Production of human tissue-type plasminogen activator in different mammalian cell lines using an Epstein-Barr virus vector.

Human tissue-type plasminogen activator (t-PA) cDNA inserted into an Epstein-Barr virus (EBV) derived expression vector was transfected into human HeLa, 293, K-562 and hamster CHO-K1 cells and the expression of t-PA was studied. The best t-PA producing cell clones were found among CHO-K1 cells (up to 11 micrograms d-1 per 10(6) cells). However, HeLa and 293 cells were most efficiently transfected, e.g. about 70% of the selected cell clones were t-PA positive. The vector DNA copy numbers correlated with the mRNA levels and the protein levels for all cell lines analysed, with the exception for the K-562 cell line, where the production of t-PA was very low. The results obtained indicated that the highest expression levels were achieved in low density cultures.


Characterization of a temperate actinophage, MPphiWR-1, capable of infecting Micromonospora purpurea ATCC 15835.

A temperate actinophage was isolated from soil using the gentamicin-producing microorganism, Micromonospora purpurea ATCC 15835 as host. The characterization of the phage represents the initial step in its development as a cloning vector. The phage isolated, MPphiWR-1, formed red- to purple-pigmented turbid plaques. Cells isolated from these plaques were resistant to superinfection with lytic mutants of MPphiWR-1. Southern blots of genomic DNA from a resistant culture showed that MPphiWR-1 integrated into the host genome. The phage was UV- or Mitomycin C-inducible. The integration, resistance to superinfection and inducibility indicated a lysogenic relationship with the host. Using MPphiE-RCPM, a lytic derivative, the phage host range was demonstrated to include members of three genera: one species each of Ampullariella and Catellatospora, and 12 species of Micromonospora. The phage belonged to Ackerman's B1 morphotype having an isometric head and a flexible noncontractile tail. The density of the phage was 1.525 g/cc. Restriction site mapping demonstrated that the phage DNA was 57.9 kb long and had cohesive ends. Using EDTA enrichment, viable mutants with deletions of at least 3.5 kb were isolated and mapped. Phage adsorption, sensitivities and plating efficiency were investigated. Non-liposome PEG-mediated transfection was demonstrated.


Micro-organisms as fertilizers and pest control agents in agricultural crops.

Micro-organisms can potentially supplement or replace the use of chemical fertilizers and pesticides in many agricultural cropping systems. Their increased use may reduce the need for nitrogen and phosphorus fertilizers, particularly in legume and cereal crops, and provide a more ecologically sound method of controlling pests and phytopathogens. Recombinant DNA technology offers the potential to extend and improve these applications. However, genetically modified micro-organisms may occasionally themselves pose environmental risks. Model ecosystems such as intact soil-core microcosms may be useful for both testing efficacy and evaluating environmental risk prior to proceeding with field trials.


Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium.

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human beta-interferon (beta-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.


A human hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by Pseudomonas aeruginosa exotoxin A.

By fusing a human hybridoma producing an IgG2 kappa antibody against human A431 epidermoid carcinoma cells with an Epstein-Barr virus-transformed human B lymphocyte producing an IgG2 kappa antibody against Pseudomonas aeruginosa exotoxin A, we established a hybrid hybridoma producing a bispecific monoclonal antibody reacting with both A431 cells and the exotoxin. Human IgG was purified from the culture supernatant of the hybrid hybridoma, and the bispecific monoclonal antibody in the IgG preparation was further separated from the two parental antibodies by hydroxyapatite high-performance liquid chromatography. The human bispecific monoclonal antibody thus obtained efficiently targeted the antibody-reactive cells, A431, for attack by the exotoxin in vitro.


Improved production of heterologous protein from Streptomyces lividans.

Protein-secreting procaryotic host organisms are currently being sought as alternatives to Escherichia coli for recombinant processing. In this study we examined how manipulation of the cultivation conditions can enhance heterologous protein production by Streptomyces lividans. The recombinant S. lividans used in this study expressed and excreted a Flavobacterium enzyme capable of hydrolyzing organophosphates. Initial shake-flask studies demonstrated that supplementing Luria-Bertani medium with moderate amounts of glucose (30 g/l), led to improved enzyme production. In fermentor studies with controlled pH, a further twofold increase in production was observed when glucose was fed continuously as compared to batch cultivation. This improved production in the glucose-fed culture may be related to a reduced accumulation of acids. Continuous feeding of both glucose and tryptone led to a further sixfold increase in production. In addition to enhancing production 25-fold, the efficiency of enzyme production and the specific activity of the excreted enzyme were also improved by glucose and tryptone feeding. These results demonstrate that in addition to genetic manipulations, optimization of cultivation conditions can lead to significant improvements in the production of heterologous proteins from Streptomyces.