Separation Of Recombinant Human Vasoactive Intestinal Peptide-Humanin Fusion Protein Expressed By E.Coli BL21(DE3)

Both vasoactive intestinal peptide (VIP) and humanin (HN) can provide neuroprotection against β-amyloid toxicity and are believed to be beneficial in the treatment of Alzheimer’s disease. In this study, we want to tandem co-express the recombinant human VIP and HN through gene engineering technology, and then find a best approach for separating and renaturing the fused proteins by Ni2+chelating resin chromatography.The rhVIP-HN gene is amplified by PCR, then the PCR product is cloned into vector pET28a(+). The recombinant pET28a(+)-VIP-HN plasmid is transformed into E.coli BL21(DE3). For expression about3h after the inoculation, the OD600of the cells reaches about0.6-0.7, and IPTG is added at a final concentration of lmmol/L to induce the expression of His6-rhVIP-HN fusion protein and following the fermentation is continued for more6h at37℃.It is known that fusion proteins expressed by the pET28a(+) vector usually form inclusion bodies. Therefore, the purification of active target proteins requires strict denaturation, renaturation and separation processes. Immobilized metal ion affinity chromatography is an efficient tool for the separation of proteins with His6tags.We design three approaches for separation:the first approach is dilution of the denatured protein into renaturation buffer followed by separation with Ni2+chelating resin. The second approach is to separate the denatured protein followed by dialysis for renaturation. And the third approach is renaturation and separation simutanelously. Through calculating the weight of the final product and analyzing the purity by gel scan based on SDS-PAGE. The yield of rhVIP-HN that is separated through Approach Ⅲ is46mg target protein/g inclusion body with purity of90%.For characterization of the target protein, MALDI-TOF-TOF MS/MS is performed according to the regulation routine. The molecular masses of the four captured peptides are almost the same as the theoretical masses and they are just within the range of the tolerance and these results indicate that the target protein is the desired rhVIP-HN.In short, the rhVIP-HN fusion protein is successfully prepared and charactered. Compared with the other two approaches, Approach III is the most suitable one for large-scale production of rhVIP-HN, which is more time-saving, buffer-saving and easier to operate.