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 R action 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. (C) 1995 Academic Press, Inc.

Battistoni, A., Mazzetti, A., Petruzzelli, R., Muramatsu, M., Federici, G., Ricci, G., et al. (1995). Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli. PROTEIN EXPRESSION AND PURIFICATION, 6(5), 579-587 [10.1006/prep.1995.1076].

Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli

BATTISTONI, ANDREA;MAZZETTI, ANNA PAOLA;FEDERICI, GIORGIO;RICCI, GIORGIO;LO BELLO, MARIO
1995

Abstract

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 R action 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. (C) 1995 Academic Press, Inc.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10
eng
Con Impact Factor ISI
chaperone; chemotactic factor; glutathione transferase; growth promotor; growth regulated protein; PelB protein; polysaccharide lyase; Polysaccharide Lyases; recombinant protein; amino acid sequence; article; biosynthesis; chemistry; cytoplasm; enzymology; Escherichia coli; genetics; human; metabolism; molecular genetics; nucleotide sequence; placenta; polyacrylamide gel electrophoresis; Amino Acid Sequence; Base Sequence; Chemotactic Factors; Cytoplasm; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Glutathione Transferase; Growth Substances; Human; Molecular Chaperones; Molecular Sequence Data; Placenta; Polysaccharide-Lyases; Recombinant Proteins; Support, Non-U.S. Gov't
Battistoni, A., Mazzetti, A., Petruzzelli, R., Muramatsu, M., Federici, G., Ricci, G., et al. (1995). Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli. PROTEIN EXPRESSION AND PURIFICATION, 6(5), 579-587 [10.1006/prep.1995.1076].
Battistoni, A; Mazzetti, A.P; Petruzzelli, R; Muramatsu, M; Federici, G; Ricci, G; Lo Bello, M
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/55182
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