Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli.
An expression vector yielding large amounts of GST P1-1 in the cytoplasm of Escherichia coli was constructed. The recombinant enzyme, obtained after purification, was characterized in its physicochemical and kinetic properties and appeared to be indistinguishable from that purified from human placenta. However, N-terminal amino acid sequencing revealed that about 50% of the recombinant GST still contained methionine as the N-terminal amino acid. Such an incomplete processing was not simply due to overproduction of GST. In fact, under growth conditions that lead to a sharp decrease in the production of the protein the N-terminal 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 chaperonins GroEL and GroES, a fraction of GST was exported into the periplasmic space with the correct N-terminal sequence. The yield of correctly 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 be obtained by genetic engineering technology.
Department of Biology, University of Rome Tor Vergata, Italy.
A
A
Battistoni
A P
AP
Mazzetti
R
R
Petruzzelli
M
M
Muramatsu
G
G
Federici
G
G
Ricci
M
M
Lo Bello
D000595:Amino Acid Sequence; D001483:Base Sequence; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D011133:Polysaccharide-Lyases / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D011133:Polysaccharide-Lyases; C074124:pectin lyase B