Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli

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The C5aR antagonist (C5aRA)(1), which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli. To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37 degrees C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496 mg/L of NusA-fused C5aRA could be produced. (c) 2013 Elsevier Inc. All rights reserved.
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Issue Date
2013-06
Language
English
Article Type
Article
Keywords

EXPRESSION; ANAPHYLATOXINS; COMPLEMENT; PROTEINS; DISEASE; SYSTEM

Citation

PROTEIN EXPRESSION AND PURIFICATION, v.89, no.2, pp.169 - 174

ISSN
1046-5928
DOI
10.1016/j.pep.2013.03.004
URI
http://hdl.handle.net/10203/174017
Appears in Collection
CBE-Journal Papers(저널논문)
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