Expressions Of HCMV Fusion Gene In E.coli Strain

With the rapid development of protein structure and function studies, big amount of purified soluble protein was necessary in related researches. Prokaryotic expression systems were the most simple and economical in obtaining the recombinant protein products that would meet the needs, while the lack of post-translational modification systems in these prokaryotes usually caused the inclusion body forms with no biological activity. Thus, it became the principle problem to attain soluble and stable protein products in prokaryotic expression systems.In this study, the fusion gene of Envelope phosphoprotein 65362-504 and Envelope glycoprotein B607-621 was obtained using Overlap extension PCR. The resulted transgene expression cassette was then subcloned into the prokaryotic vectors to give pET21b(+), pTYB11, pPAL7, pCW-ori and pMAL-c2X, respectively. By inducing with IPTG at series of temperature, we olny obtained insoluble inclusion bodies of the recombinant proteins in the transformed E. coli strains (with the expression vectors of pET21b(+), pTYB11 and pPAL7. While in the transformed strain with pCW-ori expression vector, souble protein product was obtained, and proved by SDS-PAGE and Western Blot analysis. Further, we purified the recombinant protein by using Ni-affinity chromatography, Q-FF anion-exchange chromatography and Superdex 75 gel filtration chromatography. Two bands of the protein was recovered and analyzed by MS technology, and the molecular weight were found to be 12.856 KD and 14.4 KDa, respectively, which were different from the expected protein product. In the transformed strain with pMAL-c2X, the recombinant fusion protein was found in supernatant after IPTG induce. By purifying with the Amylose affinity chromatography and digested with Factor Xa, the product was hardly detected.The results showed that the pET21b(+), pTYB11, pPAL7, pCW-ori and pMAL-c2X expression vectors were not suitable for the souble expression of pp65362-5O4-gB607-621 fusion protein in prokaryotic system. In order to obtain the souble products, we suggest the pp65362-504-gB607-621 fusion protein could be expressed in periplasm or secreting extracell of the prokaryote cells in further studies.