Peter Buckel

High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product

We have observed that proteins, such as human tissue-type plasminogen activator, pro-urokinase or gp41 of human immunodeficiency virus, which have a high content of rare codons in their respective genes, are not readily expressed in Escherichia coli. Furthermore induction of these heterologous genes leads to growth inhibition and plasmid instability. Supplementation with tRNA(AGA/AGG(Arg)) by cotransfection with the dnaY gene, which supplies this minor tRNA, resulted in high-level production with greatly improved cell viability and plasmid stability.

Control of formation of active soluble or inactive insoluble baker's yeast α-glucosidase PI in Escherichia coli by induction and growth conditions

SummaryUsing standard growth conditions (LB medium, 37°C, induction with 5 mM IPTG) yeast α-glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated α-glucosidase granules in Escherichia coli. Under these conditions active soluble α-glucosidase amounts to less than 1% of the heterologously produced protein. However, the amount of soluble active α-glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced. This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactosepermease deficient host strain containing the lacIqrepressor gene on an R-plasmid. The formation of active soluble α-glucosidase was almost 100% when E. coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e. at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.

Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure

By linking an expression cassette for human tissue-type plasminogen activator (t-PA) to an amplifiable marker gene, its introduction into Chinese hamster ovary dhfr- cells and subsequent amplification with methotrexate, we have generated cell lines that overproduce the heterologous protein and contain 300-1100 copies of the expression constructs integrated into the hamster genome. We present a detailed investigation of the fate of amplified sequences in the presence and absence of selective pressure by parallel examination of three producer cell lines with respect to relevant parameters. These include the determination of t-PA production upon continuous propagation in culture, the genomic organization of the integrated expression constructs by Southern blotting, and the localization of homogeneously staining regions by in-situ hybridization with biotinylated probes and visualization by interference reflection microscopy. We conclude that in the three cell lines examined, the decrease in production of t-PA in the absence of methotrexate selection is accompanied by decreases in the number of integrated expression constructs and the size of the amplified regions, whereas all these parameters are stable when selective pressure is maintained. The instability is probably due to the head-to-tail mode of integration of the expression constructs in the hamster genome, which increases the frequency of homologous recombination between the integrated plasmids in recombination-proficient cells in the absence of selective pressure.

Alteration of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin.

SummarySpontaneous and ethylmethane-sulfonate induced mutants of Escherichia coli resistant to gentamicin sulfate were isolated and investigated for alterations in the ribosomal protein pattern. It was found by two-dimensional polyacrylamide gel electrophoresis that three independently isolated strains did not show any spot for ribosomal protein L6. On cochromatography of radioactively labelled mutant and wild-type ribosomal proteins on carboxymethyl-cellulose columns a shift of the elution position of protein L6 was observed, the new elution positions being characteristic for the individual mutants analyzed which indicates that they possess different alterations in the L6 primary structure.Genetic analysis showed that the gentamicin resistant strains contain at least two mutations. One of them correlates with the altered L6 protein and causes an increased minimal inhibitory concentration of the drug by about 5 to 10-fold. The other mutation is not yet biochemically characterized. Its presence is connected with an about 10 to 20-fold increase in the resistance. Both mutations, when put together, confer resistance to 50 to 100 μg/ml of the antibiotic in a low salt rich medium and to 1 mg/ml in a defined medium with a high concentration of phosphate. Cross-resistance analysis demonstrated that the three gentamicin-resistant (double-mutant) strains with the altered L6 protein are resistant to 50–100 μg per ml of all other aminoglycoside antibioties tested. This forms a sharp contrast to the streptomycin resistance mutations present in strA1, strA40 or strA60 mutants which do not confer markedly increased levels of resistance to most of the other aminoglycosides.

Production of thermostable, recombinant α-galactosidase suitable for raffinose elimination from sugar beet syrup

Abstract Bacillus stearothermophilus strain KVE39, isolated from Icelandic hot springs, was shown to produce two isoenzymes of α-galactosidase and β-galactosidase. The gene for the major α-galactosidase (agaA) was cloned and expressed in E. coli and the gene product was demonstrated to possess excellent properties for application in D-raffinose removal from low green sugar beet syrup: (a) its activity is not inhibited by D-galactose or sucrose; (b) it is highly stable under process conditions; (c) its pH optimum of activity is close to neutrality. The agaA gene is constitutively expressed in E. coli at a high level: heating of crude extracts from the agaA plasmid-carrying strain and removal of precipitated host proteins by centrifugation results in simple and efficient purification.

Recombinant proteins for therapy

Recombinant therapeutic proteins have become increasingly important over the past ten years. Numerous products derived from 20 different proteins are already on the market. In this review Peter Buckel discusses the issues surrounding the use of recombinant proteins as therapeutic agents. The first generation proteins for therapy all occur naturally in humans. Protein engineering has brought forth a second generation of products with application-specific properties obtained by fusion, mutation or deletion. The third generation of therapeutic proteins is produced by patients themselves after transfer of the relevant genes. The first successful applications of this gene therapy represent a new milestone in medicine.

Expression of Pseudomonas fluorescens D-galactose dehydrogenase in E. coli

To investigate the heterologous expression of Pseudomonas genes in Escherichia coli we have cloned P. fluorescens DNA in an E. coli [cosmid] system. A colony bank representing the whole P. fluorescens chromosome was screened immunologically using a modification of the method described by Broome and Gilbert (1978). Radioactive labelling of the antibodies was replaced by conjugation with horseradish peroxidase. Among 523 E. coli colonies one was D-galactose dehydrogenase-positive. The expression of this enzyme in primary clones was lower than in the uninduced Pseudomonas. Subcloning of the D-galactose dehydrogenase gene, in vitro mutagenesis of the DNA, and coupling to a strong E. coli promoter yielded an E. coli strain that produces 90 times more of the enzyme than the induced P. fluorescens.

Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells

We report here for the first time reconstitution and secretion of functionally active antibody in non-lymphoid cells. Expression vectors for the light and the heavy chain of a monoclonal antibody directed against creatine kinase (EC 2.7.3.2) were introduced into COS and CHO Chinese hamster ovary dhfr- cells. Introduction of the expression vectors separately gave rise to immuno-reactive material in the culture supernatants, but only cotransfection of the expression plasmids resulted in secretion of protein with immuno-reactivity against antibodies directed against mouse heavy and light chains as well as specific antigen-binding affinity, as determined by enzyme-linked immunosorbent assay. Secreted kappa and gamma chains from reconstituted antibody were characterized by immunoadsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In COS cells, reconstituted antibody was transiently secreted; cotransfection of kappa and gamma chain expression plasmids with a dihydrofolate reductase (DHFR)-expression plasmid into CHO dhfr- cells gave rise to stable transformants secreting functionally active antibody.

Altered S5 and S20 ribosomal proteins in revertants of an alanyl-tRNA synthetase mutant of Escherichia coli

Summary120 phenotypic revertants of a temperature-sensitive alanyl-tRNA synthetase (alaS) mutant of Escherichia coli were isolated and screened for the ribosomal suppressor phenotype reported recently. About 20% of the mutants showed altered ribosomal sedimentation patterns which indicated a defect in ribosome assembly. The mutants were analysed by two-dimensional polyacrylamide gel electrophoresis and immunological methods for changes in ribosomal proteins.Alterations of ribosomal proteins could be identified in three mutants. One of them (0–1) had an alteration in protein S5. The mutated protein was immunologically different not only from wild type S5 but also from the altered S5 of spectinomycin resistant mutants and from the altered S5 of mutants suppressing streptomycin dependence. Two other mutants (39-1 and 64-2) had an altered S20 protein. In one of them (64-2) protein S20 was present in a reduced amount compared to the amount of S20 in wild type ribosomes.For the mutant (0–1) with the altered protein S5 a clear correlation could be demonstrated between the presence of the altered ribosomal protein and the suppression of temperature-sensitive growth of the alaS mutant.

High-throughput Functional Genomics Identifies Genes That Ameliorate Toxicity Due to Oxidative Stress in Neuronal HT-22 Cells GFPT2 Protects Cells Against Peroxide

We describe a novel genetic screen that is performed by transfecting every individual clone of an expression clone collection into a separate population of cells in a high-throughput mode. We combined high-throughput functional genomics with experimental validation to discover human genes that ameliorate cytotoxic responses of neuronal HT-22 cells upon exposure to oxidative stress. A collection of 5,000 human cDNAs in mammalian expression vectors were individually transfected into HT-22 cells, which were then exposed to H2O2. Five genes were found that are known to be involved in pathways of detoxification of peroxide (catalase, glutathione peroxidase-1, peroxiredoxin-1, peroxiredoxin-5, and nuclear factor erythroid-derived 2-like 2). The presence of those genes in our “hit list” validates our screening platform. In addition, a set of candidate genes was found that has not been previously described as involved in detoxification of peroxide. One of these genes, which was consistently found to reduce H2O2 -induced toxicity in HT-22, was GFPT2. This gene is expressed at significant levels in the central nervous system (CNS) and encodes glutamine-fructose-6-phosphate transaminase (GFPT) 2, a rate-limiting enzyme in hexosamine biosynthesis. GFPT has recently also been shown to ameliorate the toxicity of methylmercury in Saccharomyces cerevisiae. Methylmercury causes neuronal cell death in part by protein modification as well as enhancing the production of reactive oxygen species (ROS). The protective effect of GFPT2 against H2O2 toxicity in neuronal HT-22 cells may be similar to its protection against methylmercury in yeast. Thus, GFPT appears to be conserved among yeast and men as a critical target of methylmercury and ROS-induced cytotoxicity.