Enhanced recombinant insulin production in transgenic Escherichia coli that heterologously expresses carrot heat shock protein 70

Abstract

Abstract Recombinant insulin production was enhanced in transgenic Escherichia coli (E. coli BL21, DE3) that heterologously expressed carrot (Daucus carota L.) heat shock protein 70 (DcHsp70). The gene encoding DcHsp70 with the constitutive bacterial lipoprotein promoter was inserted into the E. coli genome using lambda Red-mediated homologous recombination. Nucleotide sequences for the human proinsulin, insulin B and insulin A chains were separately cloned into the pVFT 2S expression vector containing a 6xhistidine and glutathione S-transferase (GST) tag. When induced by isopropyl β- d -1-thiogalactopyranoside treatment, the expression levels of the proinsulin and insulin A and B chain fusion proteins were higher in the transgenic cell lines that heterologously expressed DcHsp70 than in the control cell line. The 6xHis-GST tag was removed from the three versions of the fusion proteins by tobacco etch virus protease. As an active molecular chaperone, DcHsp70 could increase recombinant insulin production when heterologously expressed in E. coli. Our results suggest a possible use of plant Hsps as effective molecular chaperones to increase recombinant protein production in E. coli.