Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I.
Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent.
Canova-Davis
E
E
Department of Medicinal and Analytical Chemistry, Genentech Inc., South San Francisco, CA 94080.
Eng
M
M
Mukku
V
V
Reifsnyder
D H
DH
Olson
C V
CV
Ling
V T
VT
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D006898:Hydroxylamines; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D019811:Hydroxylamine; D007334:Insulin-Like Growth Factor I; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase