Cloning And Expression Of DNA Sequences Encoding Human Intact And Truncated Mature Peptides Of Insulin-like Growth Factor Ⅰ

Human insulin-like growth factor-1 (IGF-1) is a basic polypeptide containing 70 amino acid residues. Its truncated form, i.e. des(l-3)IGF-1, is three residues shorter than the intact form at N- terminal. IGF-1 has many biological actions such as mitogenic, metabolic effects and others. It has important role on bone remodeling, fetation and nerve development. Its medical uses of curing diabetes mellitus, dawfism and osteoporosis are just developing.To obtain enough IGF-1 for basic or clinic research, we must choice an convenient method other than extraction from human materials, eg. plasma or others. In this article, the author amplified DNA sequences encoding the mature peptides of human intact and truncated IGF-1 gene from human genomic DNA by the new way of alternative PCR established and named as exons-piecing together method by this laboratory. The IGF-1 and des(l-3)IGF-l gene were cloned in PGEM-T vector and then subcloned in expression plasmid PGEX-3X. Two recombinant constructs known as pGEX-3X-IGF-l and pGEX-3X-des(l-3)IGF-l were transformed to E.coli DH5 a respectively. Having been induced by isopropyl thio- $ -D-galactopyranoside( IPTG), the host bacteria did express the 32KD (molecular weight) GST-IGF-1 and GST-des(l-3)IGF-l fusion protein identified by SDS-PAGE. The expression level was 5mg GST fusion protein per liter bacteria culture. Western Blotting indicated that theexpression protein had the immunoreactivity of IGF-1. Then after purification, renaturation, cleavage and ultrafiltration centrifugation, IGF-1 and des(l-3)IGF-l protein were obtained. The biological activity of expressed products were examined with thiazoyl blue tetrazolium bromide(MTT) method, and the myoblast as target cell. It concluded that the biological activity of des(l-3)IGF-l protein was 1.5 to 4 times higher than that of IGF-1 form.