TY - JOUR
T1 - proBA complementation of an auxotrophic E. coli strain improves plasmid stability and expression yield during fermenter production of a recombinant antibody fragment
AU - Fiedler, Markus
AU - Skerra, Arne
PY - 2001/8/22
Y1 - 2001/8/22
N2 - 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.
AB - 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.
KW - E. coli expression
KW - Immunoglobulin
KW - Metabolic selection
KW - Monoclonal antibody IN-1
KW - Protein engineering
UR - http://www.scopus.com/inward/record.url?scp=0035934280&partnerID=8YFLogxK
U2 - 10.1016/S0378-1119(01)00629-1
DO - 10.1016/S0378-1119(01)00629-1
M3 - Article
C2 - 11675003
AN - SCOPUS:0035934280
SN - 0378-1119
VL - 274
SP - 111
EP - 118
JO - Gene
JF - Gene
IS - 1-2
ER -