title: proBA complementation of an auxotrophic E. coli strain improves plasmid stability and expression yield during fermenter production of a recombinant antibody fragment.
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 F(ab) 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 Delta proBA 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 F(ab) 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.
authors.affiliation: Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Fiedler
authors.forename: A
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D005819:Genetic Markers / Q000235:genetics; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D009876:Operon / Q000235:genetics*; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D003470:Culture Media; D005819:Genetic Markers; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase