Abstract:

Abstract:Objective To clone fibroblast growth factor-21(FGF21)［Arg59］ mutant gene, express it in E.coli and purify the expression products, and provide basis   for Pull-down test and further study on human hepatoma in mouse models.  Methods The codon of the 59th Lysine of FGF21 cDNA was replaced by Arginine codon by PCR site-directed mutation. Then the mutant fragment and SUMO fragment were fused by PCR and subcloned into pET20b expression vector to obtain recombinant plasmid pET20b-SUMO-FGF21［Arg59］ . The recombinant plasmid was transformed into E.coil BL21 (DE3) and the  expression was induced by IPTG. The expressed protein was purified by Ni- NTA Agarose, SUMO protease cutting, molecular sieve chromatography and so on.  Results The 546 bp gene fragment was amplified by PCR，and the sequencing result showed there was an aim mutation. The positive clones of pET20b-SUMO-FGF21［Arg59］ was identified by PCR and digestion identification. A  fusion protein whose relative molecular mass was 31 500 was soluably expressed after induction.The  Western blotting result indicated that the purified product was mature FGF21［Arg59］ mutant protein.  Conclusion FGF21［Arg59］ mutant gene is cloned and the fusion gene is expressed in E.coil and purified successfully.

Key words: fibroblast growth factor-21, mutant, SUMO fusion expression, purification