Expression and purification of human insulin-like growth factor-I

Human insulin-like growth factor Ⅰ(HIGF-Ⅰ: also called Somatomedine C) is a 70 aa protein found in human plasma. It has been shown to promote growth and differentiation of various cell types. It has also insulin-like hypoglycemic activity. Therefore, IGF-Ⅰ may be useful in the therapies of dwarfism, osteoporosis and insulin-resistant diebetes. Biological and pharmacological studies of IGF-Ⅰhas been restricted by extreme insufficiency of the purified samples. It has been purified from human plasma. But several tons of human plasma yielded only a few miligrams of the peptoed hormone. Moreover, the purified sample obtained by chemical synthesis was quite small. Thus, IGF-Ⅰ gene which was isolated from humsn adult liver cDNA libraroes, has been expressed in E. coli system using recombinant DNA techniques. Nature form of IGF-Ⅰ cDNA was fused with a truncated lacZ gene, using synthetic liker containing enterokinase or hydroxylamine cleavage site. In expressed cell lysate, β-galactosidase/IGF-Ⅰfusin protein was about 30% of the total cellular proteins. The fusion proteins which were purified with Sephacryl S-200 column chromatography were treated with enterokinase or hydroxylamine. The released IGF-Ⅰ was purified with SP-Sephadex C-25 column chromatography. The purified IGF-Ⅰ was further purified using reverse-phased HPLC. The amino acid sequence of the purified recmbinant IGF-Ⅰ was identcal with that of native IGF-Ⅰ. Growth promoting activity of IGF-Ⅰ was confirmed from(³H)-thymidine incorporation in L(low 6)-myoblast. For the development of sensitive and accurate method of detection IGF-Ⅰ concentrations in blood system. several monoclonal antibodies recognizing different epitopes are generated. Two types of monoclonal antibodies(5C9 and 1B3) recognizing different epitopes were selected by epitope mapping experiment. The development of ELISA system using selected monoclonal antibodies for IGF-Ⅰ made it possible to measure IGF-Ⅰ concentrations in blood.과기처에서 시행한 일반출연 연구사업의 연구보고서위탁연구과제 1: 한방면역 증강제와 IGF-I의 면역증진 비교연구 / 대전대학교 한의학 대학교 ; 김성훈 연구책