CYTOPLASMIC AND PERIPLASMIC PRODUCTION OF HUMAN PLACENTAL GLUTATHIONETRANSFERASE IN ESCHERICHIA-COLI

An expression vector yielding large amounts of GST P1-1 in the cytopla sm of Escherichia coli was constructed. The recombinant enzyme, obtain ed after purification, was characterized in its physicochemical and ki netic properties and appeared to be indistinguishable from that purifi ed from human placenta. However, N-terminal amino acid sequencing reve aled that about 50% of the recombinant GST still contained methionine as the N-terminal amino acid. Such an incomplete processing was not si mply due to overproduction of GST. In fact, under growth conditions th at lead to a sharp decrease in the production of the protein the N-ter minal methionine was not removed. GST was unable to translocate across the bacterial membrane when it was fused to the leader peptide of the pelB gene from Erwinia carotovora and accumulated in the cytoplasm in a soluble and active conformation. However, when this fusion protein was produced in a bacterial strain overexpressing the bacterial chaper onins GroEL and GroES, a R action of GST was exported into the peripla smic space with the correct N-terminal sequence. The yield of correctl y processed GST accounted for 12% of total GST present in the E. coli cells. Our results suggest that chaperonins are able to interact with nascent GST, thus maintaining the protein in an export-competent form and that E. coli strains with enhanced secretory characteristics may b e obtained by genetic engineering technology. (C) 1995 Academic Press, Inc.