Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies.
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.
Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712-0231, USA.
Tomohiro
T
Makino
Georgios
G
Skretas
Tae-Hyun
TH
Kang
George
G
Georgiou
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D057166:High-Throughput Screening Assays; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods
D029968:Escherichia coli Proteins; D007074:Immunoglobulin G