title: High-level expression of alpha-human atrial natriuretic peptide from multiple joined genes in Escherichia coli.
abstract: A method is described which allows alpha-human atrial natriuretic peptide to be synthesized in stable form and with high yield in Escherichia coli. In the final expression system, eight copies of the synthetic alpha-hANP gene were linked in tandem, separated by codons specifying a 4-amino-acid (aa) linker with lysine residues flanking the authentic N and C termini of the 28-aa hormone. This sequence was in turn joined to the 3' end of a fragment containing the lac promoter and a leader sequence coding for the first seven N-terminal amino acids of beta-galactosidase. The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was easily purified by urea extraction of the insoluble cell fraction. The purified protein was cleaved into monomers by digestion with endoproteinase Lys-C, trimmed to expose the authentic C terminus by digestion with carboxypeptidase-B and a single disulfide bond was formed by gentle oxidation with potassium ferricyanide. The fully processed recombinant peptide was shown by reverse phase liquid chromatography to be indistinguishable from the chemically synthesized standard alpha-hANP in both the reduced and in the folded form.
authors.affiliation: Molecular Genetics Group, Connaught Research Institute, Willowdale, Ontario, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Lennick
authors.forename: J R
authors.initials: JR
authors.lastname: Haynes
authors.forename: S H
authors.initials: SH
authors.lastname: Shen
mesh_terms: D009320:Atrial Natriuretic Factor / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D005786:Gene Expression Regulation; D006801:Humans; D007763:Lac Operon; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D009320:Atrial Natriuretic Factor