Synthesization, expression, purification and activity assay of hIGF-1 / 中国病理生理杂志

ABSTRACT

AIM: To express the synthesized human insul in like growth factor I (hIGF-1) gene in E.coli with high expression level a nd explore the way to increase the efficiency of factor Xa cleavage. METHODS: The gene of hIGF-1 was designed and synthesized accordi n g to the preference of E.coli. A fusion protein with a recognized site of fa ctor Xa between CBD (cellulose binding domain) and hIGF-1 was expressed and puri fied by cellulose affinity chromatography. MTT method was used to assay the bioa ctivity of CBD-IGF fusion protein. hIGF-1 was released by factor Xa. In order to improve the sensitivity of fusion protein to factor Xa, the short flexible pept ide (Gly-Thr-Gly- Gly-Gly-Ser-Gly) was added before the recognized site of fac tor Xa. RESULTS: SDS-PAGE results indicated that the CBD-IGF fusion prot ein was expressed and purified . Biological assay results indicated CBD-IGF fusi on protein could promote the growth of NIH3T3 cell. The short flexible peptide (Gly-Thr-Gly-Gly-Gly-Ser-Gly), which was added before the recognized site of f actor Xa, improved the sensitivity of fusion protein to factor Xa. CONCLUSION: CBD-IGF fusion protein with bioactivite are expresse d and purified. The amio acid sequences changes between the site recognize of fa ctor Xa can help to improve the cleavage efficiency of Factor Xa.