A plasmid system for optimization of Fab' production in Escherichia coli: importance of balance of heavy chain and light chain synthesis.
We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.
Humphreys
David P
DP
Celltech R+D, 216 Bath Road, Slough, SL1 4EN, Berkshire, UK. dhumphre@celltech.co.uk
Carrington
Bruce
B
Bowering
Leigh C
LC
Ganesh
Ravindra
R
Sehdev
Mukesh
M
Smith
Bryan J
BJ
King
Lloyd M
LM
Reeks
Dominic G
DG
Lawson
Alastair
A
Popplewell
Andrew G
AG
D001483:Base Sequence; D003062:Codon; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D017930:Genes, Reporter; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids
D003062:Codon; D007140:Immunoglobulin Fab Fragments