Ralf Mattes

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.

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.

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.