Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

Coexpression of two classes of folding accessory proteins, molecular chaper ones and foldases, can be expected to improve the productivity of soluble a nd active recombinant proteins. In this study, horseradish peroxidase (HRP) , which has four disulfide bonds, was selected as a model enzyme and overex pressed in Escherichia coli. The effects of coexpression of a series of fol ding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), D sbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E, coli were examined. Active HRP was produced by very mild inducti on with 1 muM isopropyl-beta -D-thiogalactopyranoside (IPTG) at 37 degreesC , whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPT G in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, c oexpression of Dsb proteins improves both the cell growth and the productiv ity of HRP.