title: Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins.
abstract: Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, 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 IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.
authors.affiliation: Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Kondo
authors.forename: J
authors.initials: J
authors.lastname: Kohda
authors.forename: Y
authors.initials: Y
authors.lastname: Endo
authors.forename: T
authors.initials: T
authors.lastname: Shiromizu
authors.forename: Y
authors.initials: Y
authors.lastname: Kurokawa
authors.forename: K
authors.initials: K
authors.lastname: Nishihara
authors.forename: H
authors.initials: H
authors.lastname: Yanagi
authors.forename: T
authors.initials: T
authors.lastname: Yura
authors.forename: H
authors.initials: H
authors.lastname: Fukuda