A plasmid system for optimization of Fab′ production in Escherichia coli:: importance of balance of heavy chain and light chain synthesis

被引：42

作者：

Humphreys, DP ^{[1
]}

Carrington, B ^{[1
]}

Bowering, LC ^{[1
]}

Ganesh, R ^{[1
]}

Sehdev, M ^{[1
]}

Smith, BJ ^{[1
]}

King, LM ^{[1
]}

Reeks, DG ^{[1
]}

Lawson, A ^{[1
]}

Popplewell, AG ^{[1
]}

机构：

[1] Celltech Ltd, R&D, Slough SL1 4EN, Berks, England

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2002年 / 26卷 / 02期

关键词：

codon usage; E coli; heterodimeric Fab '; M13 gene VIII signal peptide;

D O I：

10.1016/S1046-5928(02)00543-0

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 mg L-1 from E coli fermentations and will enable rapid variable region subcloning and selection of future Fab' expression plasmids. (C) 2002 Elsevier Science (USA). All rights reserved.

引用

页码：309 / 320

页数：12

共 17 条

[1] Therapeutic antibody fragments with prolonged in vivo half-lives [J].