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

Markus Fiedler, Arne Skerra

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

The proline-auxotrophic Escherichia coli K12 strain JM83 harbouring an expression vector providing the proBA gene in trans was utilized for the fermenter production of the partially humanized IN-1 antibody Fab fragment. Thus, plasmid-mediated complementation of the chromosomal proBA deletion was employed as a second selection mechanism, together with a chloramphenicol resistance, in order to (i) abolish plasmid loss and (ii) benefit from E. coli JM83 as an expression strain with approved periplasmic protein secretion characteristics in the presence of a minimal medium. Starting from the generic vector pASK75, which makes use of the tightly regulated and chemically inducible tet promoter for foreign gene expression, a set of new vectors carrying the entire or part of the proBA operon was constructed and compared concerning their capability of functional ΔproBA complementation as well as recombinant protein yield. As a result, the vector pMF1 was developed, where transcription of the proBA operon is controlled by its own constitutive promoter and terminator sequences, permitting the transformed JM83 strain to grow under glucose/ammonia minimal culture conditions. When pMF1 was used for the fermenter production of the IN-1 Fab fragment, no plasmid loss was observed during the growth and induction phases, and the yield of functionally purified recombinant protein was found to be considerably improved.

Original languageEnglish
Pages (from-to)111-118
Number of pages8
JournalGene
Volume274
Issue number1-2
DOIs
StatePublished - 22 Aug 2001

Keywords

  • E. coli expression
  • Immunoglobulin
  • Metabolic selection
  • Monoclonal antibody IN-1
  • Protein engineering

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