Purification of IGF-I in Bovine Colostrum and its Application for Animal Production Research.

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Insulin-like growth factor-I (IGF-I) is a biologically active peptide consisting of 70 amino acids (mol wt. 7,649). Its chemical structure is homologous to proinsulin. The primary structure of IGF-I are almost identical in mammalian species tested (human, rat, mouse, porcine, bovine, ovine).Biological action of IGF-I, firstly found as sulfation factor, has been shown by recent investigations to stimulate proliferation and differentiation of various cell types. Growth hormone stimulates the secretion of IGF-I, then IGF-I stimulates the growth of young animals through accelerating bone elongation and protein accumulation. On this point IGF-I has been recognized to be of importance for animal production. We showed that the concentration (0.5mg/l) of IGF-I in bovine colostrum exceeded that in blood plasma which has been used as a biological material to extract IGF-I, suggesting availability of colostrum to be an useful material for purifying IGF-I. Colostrum was collected on the day of par … More turition. Chymosin was added to colostrum. After incubation at 37 C, the lipid- and casein-free supernatant was obtained by centrifugation. Acid-ethanol mixture (12.5% HC1, 87.5% EtOH) was added to dissociate IGF-I from its binding protein, followed by further addition of cold acetone to get acetone powder. We used the acetone powder as starting material. The powder was dissolved in 1M acetic acid solution to apply to sephadex G-50 column. Eluate of IGF-I immunoreactive fractions, containing 0.7 ug IGF-I/mg protein, was lyophilized and gel filtration procedure by TSKG3000SW was performed. The TSK column was eluted with 1M acetic acid, IGF-I being purified to 1.6 ug/mg protein. The lyophilized sample was dissolved with 20% acetonitrile in 0.05% TFA, and applied to the first ODP-50 reverse-phase HPLC. IGF-I immunoreactivity was eluted by a two-step linear gradient to 80% acetonitrile. IGF-I concentration of the eluate was 32 ug/mg protein. IGF-I was further purified by the second ODP-50 reverse phase HPLC. The bioassay using rat myoblast subculture cells clearly showed IGF-I bioactivity in the samples eluted from Sephadex and TSK gel column. Less