Fusion Expression And Purification Of Human Vasostatin Fragment In E.coli And Its Activity Assay

Vasosatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. Present study has suggested that the antiangiogenic activities of vasostatin reside in a domain that is localized to calreticulin N-terminal amino acids 120-180. This research was designed to find out the shorter effective activity fragments of vasostatin. Using the plasmid pUC18-vasostatin contatining full-length human vasostatin cDNA as a template, four vasostatin fragments, which contatin different cDNA sequences, were amplified by polymerase chain reaction(PCR). The target cDNA were inserted into the expression vector pGEX-4T-3 respectively, thus constructed recombinant vectors pGEX-4T-3/vasostatin-A, pGEX-4T-3/vasostatin-B, pGEX-4T-3/vasostatin-C and pGEX-4T-3/vasostatin-A-C, then these recombinant vectors were transformed into E.coli BL21(DE3). The fusion proteins were expressed successfully by IPTG induction, and SDS-PAGE assays informed these fusion proteins were expressed in inclusion body form. Changing the induction condition would increase the soluble fusion proteins. The expression levels of target proteins in inclusion body reached up to 55% of the total proteins. Fusion proteins were purified by GST affinity chromatography. Vasostatin fragments were released from the fusion proteins by thrombin cleavages and tested the biological activity through MTT method. As a result, the purified vasostatin frgments can inhibit the proliferation of endothelial cells with similar bioactivity to full-length vasostatin, and the vasostatin fragments consisted of amino acids 135-164 has an obvious advantage. This suggested that antiangiogenic activities of human vasostatin resid in the domain which accessible fragment between amino acids 135-164. The search maybe presents some challenging opportunities in doing so would help foster a new era of cancer therapeutics in the clinic.