Improvement of productivity of active from of glutamate racemase in escherichia coli by coexpression of folding accessory proteins

摘要

Since two classes of folding accessory proteins, molecular chaperones and foldases, prevent the misfolding of newly synthesized polypeptides in the cell, their coexpession could be expected to improve the productivity of soluble and active recombinant proteins. Escherichia coli cytoplasmic glutamate racemase (GluR), which has five cysteine thiol groups and no disulfide bond, was selected as a model enzyme and overespressed in E.coli. The effects of coexpressing 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 GluR in E.coli were examined. A relatively large amolunt of active GluR produced by mild induction with 10 #mu#M isoporpyl-#beta#-D-thiogalactopyranoside (IPTG). Active GluR productivity was further increased 2.2-2.3-fold by coexpression of GroEL/ES,Trx, or DsbB-DsbD (DsbBD), while it was decreased by coexpression of DnaK-DnaJ-GrpE and TF. These results demonstrate that coexpression of appropriate folding accessory proteins could significantly improve the productivity of active form of proteins in E.coli.