Synthesization, expression, purification and activity assay of hIGF-1
- VernacularTitle:人类胰岛素生长因子Ⅰ基因的合成、表达与纯化
- Author:
Yuan GAO
;
Rongjie YU
;
An HONG
;
Zhiyin LI
- Publication Type:Journal Article
- Keywords:
Insulin-like growth factor 1;
Fusion proteins;
Factor Xa;
Genes;
Gene expression
- From:
Chinese Journal of Pathophysiology
1986;0(02):-
- CountryChina
- Language:Chinese
-
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.
