Expression, purification and characterization of recombinant human insulin-like growth factor I in yeast.
Insulin-like growth factor I (IGF-I) is a 70 amino acid (aa) protein that is structurally similar and functionally related to insulin. We have inserted a synthetic gene coding for human IGF-I into a Saccharomyces cerevisiae expression vector utilizing the MF alpha 1 promoter and pre-pro leader peptide. This vector directs the expression and secretion of native, biologically active growth factor. Cleavage of the pre-pro alpha factor leader sequence in vivo results in the secretion of a 70-aa recombinant IGF-I molecule with the native N-terminal glycine residue. Human IGF-I purified from yeast culture supernatant is equipotent to serum-derived IGF-I in inhibiting [125I]IGF-I binding to type-I IGF receptors and crude human serum-binding proteins. Recombinant IGF-I is also equipotent to human IGF-I in the stimulation of DNA synthesis in rat aortic smooth-muscle cells. In contrast, yeast recombinant IGF-I is less potent than serum-derived IGF-I in binding to type-2 IGF receptors. The ability to produce native, biologically active IGF-I in yeast will allow the elucidation of binding domains through the expression and characterization of specific structural analogs.
D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007334:Insulin-Like Growth Factor I / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013002:Somatomedins / Q000235:genetics*
D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I