proBA complementation of an auxotrophic E-coli strain improves plasmid stability and expression yield during fermenter production of a recombinant antibody fragment

The proline-auxotrophic Escherichia coli K12 strain JM83 harbouring an expr ession vector providing the proBA gene in trans was utilized for the fermen ter production of the partially humanized IN-1 antibody F-ab fragment. Thus , plasmid-mediated complementation of the chromosomal proBA deletion was em ployed as a second selection mechanism, together with a chloramphenicol res istance, in order to (i) abolish plasmid loss and (ii) benefit from E. coli JM83 as an expression strain with approved periplasmic protein secretion c haracteristics in the presence of a minimal medium. Starting from the gener ic vector pASK75, which makes use of the tightly regulated and chemically i nducible tet promoter for foreign gene expression, a set of new vectors car rying the entire or part of the proBA operon was constructed and compared c oncerning their capability of functional Delta proBA complementation as wel l as recombinant protein yield. As a result, the vector pMF1 was developed, where transcription of the proBA operon is controlled by its own constitut ive promoter and terminator sequences, permitting the transformed JM83 stra in to grow under glucose/ammonia minimal culture conditions. When pMF1 was used for the fermenter production of the IN-1 F-ab fragment, no plasmid los s was observed during the growth and induction phases, and the yield of fun ctionally purified recombinant protein was found to be considerably improve d. (C) 2001 Elsevier Science B.V. All rights reserved.