期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 82, 期 1, 页码 197-204出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2011.12.010
关键词
Disulfide bond; Escherichia coli; Protein disulfide isomerase; Protein expression; Single chain variable fragment
类别
资金
- Frontier Biomedical Science Underlying Organelle Network Biology, Osaka University
- Grants-in-Aid for Scientific Research [22020037, 23121532, 24510318] Funding Source: KAKEN
Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed pCold-PDI vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains. (C) 2012 Elsevier Inc. All rights reserved.
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