title: Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3, following their expression in Escherichia coli as fusion proteins.
abstract: The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.
authors.affiliation: Department of Biochemistry, University of Adelaide, South Australia.
authors.forename: R
authors.initials: R
authors.lastname: King
authors.forename: J R
authors.initials: JR
authors.lastname: Wells
authors.forename: P
authors.initials: P
authors.lastname: Krieg
authors.forename: M
authors.initials: M
authors.lastname: Snoswell
authors.forename: J
authors.initials: J
authors.lastname: Brazier
authors.forename: C J
authors.initials: CJ
authors.lastname: Bagley
authors.forename: J C
authors.initials: JC
authors.lastname: Wallace
authors.forename: F J
authors.initials: FJ
authors.lastname: Ballard
authors.forename: M
authors.initials: M
authors.lastname: Ross
authors.forename: G L
authors.initials: GL
authors.lastname: Francis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D004247:DNA