title: Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli.
abstract: The export of heterologous products into the conditioned medium of an Escherichia coli culture offers the advantages of a higher product yield, an increased probability of recovering an intact recombinant protein, proper folding for biological activity, and greater stability of a secreted product. In this report, we describe the development of an optimized direct expression system, designed to maximize the extracellular accumulation of recombinant glycine-extended salmon calcitonin peptide (sCTgly). We have used dual promoters, an ompA signal sequence, co-expression of homologous secretion factor genes, and multiple gene cartridges to express the sCTgly. High-density fermentation conditions have been developed that allow for the selective secretion and accumulation of the expressed sCTgly at very high levels. Purification and in vitro enzymatic conversion by peptidylglycine alpha-amidating monooxygenase yields authentic, biologically active salmon calcitonin. This recombinant production technology is applicable to a variety of amidated peptide hormones.
authors.affiliation: Unigene Laboratories, Inc., 110 Little Falls Road, Fairfield, NJ 07004, USA. vicki347@yahoo.com
authors.forename: Martha V L
authors.initials: MV
authors.lastname: Ray
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Meenan
authors.forename: Angelo P
authors.initials: AP
authors.lastname: Consalvo
authors.forename: Carrie A
authors.initials: CA
authors.lastname: Smith
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Parton
authors.forename: Amy M
authors.initials: AM
authors.lastname: Sturmer
authors.forename: Paul P
authors.initials: PP
authors.lastname: Shields
authors.forename: Nozer M
authors.initials: NM
authors.lastname: Mehta
mesh_terms: D001483:Base Sequence; D002116:Calcitonin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D005998:Glycine / Q000737:chemistry*; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D017077:Culture Media, Conditioned; D017931:DNA Primers; D011498:Protein Precursors; D011994:Recombinant Proteins; C028815:salmon calcitonin; D002116:Calcitonin; D005998:Glycine