期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 77, 期 1, 页码 104-111出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2010.11.016
关键词
Escherichia coli; Recombinant protein expression; Ligation-independent cloning; OsmY; DsbA; pelB
类别
资金
- Novo Nordisk
- Novo Nordisk Foundation
Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts. Here we present a screening strategy for expression of biomedically relevant proteins in Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth factor binding proteins. This rapid screening approach appears highly suitable for high-throughput efforts targeting either large sets of proteins or more focused investigations regarding individual high-profile targets. (c) 2010 Elsevier Inc. All rights reserved.
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