title: Development of an optimized refolding process for recombinant Ala-Glu-IGF-1.
abstract: Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w) was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements; (i) Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; or (ii) Cys6-Cys52, Cys18-Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.
authors.affiliation: Hagedorn Research Laboratory, Gentofte, Denmark.
authors.forename: K R
authors.initials: KR
authors.lastname: Hejnaes
authors.forename: S
authors.initials: S
authors.lastname: Bayne
authors.forename: L
authors.initials: L
authors.lastname: Nørskov
authors.forename: H H
authors.initials: HH
authors.lastname: Sørensen
authors.forename: J
authors.initials: J
authors.lastname: Thomsen
authors.forename: L
authors.initials: L
authors.lastname: Schäffer
authors.forename: A
authors.initials: A
authors.lastname: Wollmer
authors.forename: L
authors.initials: L
authors.lastname: Skriver
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000494:pharmacology; D001667:Binding, Competitive; D002942:Circular Dichroism; D003545:Cysteine; D010450:Endopeptidases / Q000235:genetics; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; C079682:insulin-like growth factor I, N-Ala-Glu-; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D010450:Endopeptidases; D000626:Aminopeptidases; D003545:Cysteine