Cloning And Expression Of Visfatin And Screening Of Oligopeptides Binding With Visfatin

Visfatin is a novel polypeptide hormone which is able to bind to r insulin receptor and mimic the physiologic effects of insulin. Actually, visfatin has insulin-like metabolic effects in vivo and in vitro therefore it becomes another hot spot in endocrinological research. However it turns out that visfain and insulin do not share a common binding site in insulin receptor. It is not certain what is its binding site in insulin receptor and whether it has its specific receptor so far.To obtain more and pure visfatin, an efficient expression system is essential. In this experiment, total RNA was extraction from visceral fat and the gene encoding for visfatin mature peptides was amplified by RT-PCR. And then the gene was inserted into cloning vector pGM-T and fusion expressing vector pQE-30Xa in turn. Furthermore, recombinant vector pQE-30Xa/visfatin was transformed into E.coli M15 [pREP4]. The target proteins containing hexahistidyl affinity tag facilitates binding to Ni-NTA resin in metal chelate affinity chromatography. After the proteins binding non-specifically to resin were removed by washing solutions with gradient increasing concentration of imidazole, the target proteins were eluted from resin with apparent higher concentration of imidazole and concentrated by centrifuge ultra filtration. The hexahistidyl affinity tag was removed by Xa and target protein was identified by SDS-PAGE and Western blot. After the culturing conditions were optimized, the yield of visfatin was 300mg/L culture medium and accounted for 46.8% of total protein in E.coli M15[pREP4]. The recombinant visfatin was identified to bind with insulin receptor by protein-protein interaction and has a dose-dependent effect.To explore the peptide sequences binding to visfatin, the purifled-protein-coated polystyrene plate was used to screen recombinant phage that could bind onto it. After three rounds of affinity screening, 24 phages that could bind specifically with visfatin were selected from a random phage-displayed seven-peptide library. The peptide sequences of the positive phage clones were analyzed and were identified by ELISA. Among these sequences, there were 8 clones of AAKTPTE, 4 clones of ATTVPAS and 4 clones of MSLQQEH. As a result, the sequence of AA(X)TPT(X) was the most frequent sequence in all of sequences analyzed. Probably AA(X)TPT(X) is the essential motif in peptides which could bind with visfatin.