大肠杆菌的综合工程改造以增强 IgG 抗体的表达。
Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies.
机构信息
Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712-0231, USA.
出版信息
Metab Eng. 2011 Mar;13(2):241-51. doi: 10.1016/j.ymben.2010.11.002. Epub 2010 Dec 3.
Abstract
The expression of IgG antibodies in Escherichia coli is of increasing interest for analytical and therapeutic applications. In this work, we describe a comprehensive and systematic approach to the development of a dicistronic expression system for enhanced IgG expression in E. coli encompassing: (i) random mutagenesis and high-throughput screening for the isolation of over-expressing strains using flow cytometry and (ii) optimization of translation initiation via the screening of libraries of synonymous codons in the 5' region of the second cistron (heavy chain). The effects of different promoters and co-expression of molecular chaperones on full-length IgG production were also investigated. The optimized system resulted in reliable expression of fully assembled IgG at yields between 1 and 4 mg/L of shake flask culture for different antibodies.
摘要
在分析和治疗应用中，大肠杆菌中 IgG 抗体的表达引起了越来越多的关注。在这项工作中，我们描述了一种全面系统的方法，用于开发二顺反子表达系统，以增强大肠杆菌中 IgG 的表达，包括：（i）随机诱变和高通量筛选，使用流式细胞术分离高表达菌株；（ii）通过筛选第二个顺反子（重链）5'区域的同义密码子文库优化翻译起始。还研究了不同启动子和共表达分子伴侣对全长 IgG 生产的影响。优化后的系统可在摇瓶培养中实现不同抗体的完全组装 IgG 的可靠表达，产量在 1 至 4mg/L 之间。
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