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

Abstract

The C5aR antagonist (C5aRA)1Abbreviations used: C5aRA, C5aR antagonist; NusA, N utilization substrate A; MBP, maltose-binding protein; TrxA, thioredoxin A; C5aR, C5a receptor; PCR, polymerase chain reaction; TB, terrific broth; NTA, ni-nitrilotriacetic acid; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffered saline; BSA, bovine serum albumin; DCW, dry cell weight; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.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°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 496mg/L of NusA-fused C5aRA could be produced.