Comparison of two chemical cleavage methods for preparation of a truncated form of recombinant human insulin-like growth factor I from a secreted fusion protein.
We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage.
Forsberg
G
G
KubiGen AB, Stockholm, Sweden.
Baastrup
B
B
Brobjer
M
M
Lake
M
M
Jörnvall
H
H
Hartmanis
M
M
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000235:genetics; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008715:Methionine / Q000032:analysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010957:Plasmids; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics; D013002:Somatomedins / Q000032:analysis*; D014364:Tryptophan / Q000032:analysis
D011993:Recombinant Fusion Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D014364:Tryptophan; D008715:Methionine