title: Expression of honeybee prepromelittin as a fusion protein in Escherichia coli.
abstract: Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.
authors.affiliation: Department of Surgery, St. George's Hospital Medical School, London, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: He
authors.forename: I
authors.initials: I
authors.lastname: Adcock
authors.forename: D
authors.initials: D
authors.lastname: Chapman
authors.forename: J
authors.initials: J
authors.lastname: Lucy
authors.forename: B
authors.initials: B
authors.lastname: Austen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001516:Bees / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008555:Melitten / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D008555:Melitten; C016694:prepromelittin; D004247:DNA; D001616:beta-Galactosidase