Human Heat Shock Protein GP96 Gene Clone, Expression And Purification In E.Coli

The gp96 is the endoplasmic recticulum (ER)paralogue of heat shock protein 90, and it is an abundant protein of the ER lumen. As a chaperone, gp96 can help some proteinsto refold or reassamble in vivo. The most important feature of gp96 is that its peptide binding complex can access the cross-priming pathways of antigen presenting cells and the bound peptides can be represented onto MHC class I molecules to elicit CD8+T cell activation. Generally it is essential to obtain enough amounts of pure gp96 to meet the needs of study and application.Gp96 preparations derived from cancer cells and virus-infected cells can have been shown previously to elicit cancer-specific or virus-specific immunity. The immunogenicity of gp96 preparations have been attributed to peptides associated with it. But gp96 is generally expressed at low level in cells. Gp96 preparations from limited cells or tissue are difficult to meet the needs of study and application. So our current study aims to obtain enough amounts of pure human gp96 in vitro. In our study, human gp96 gene was obtained by RT-PCR .then cloned into pET30-a(+) vector,and expressed the recombinant in E.coli Blstar. Gp96 was expressed with soluble forms. But the recombinant gp96 in E. coli is easy to degrade and formaggregates. Through ultra centrifugation, ammonium sulfate precipitation. And after purification by Ni-affinity column, ConA Sepharose affinity chromatography, anion exchange column Gel-filtration, most contaminated fragments and aggregates were removed and certain amount of soluble gp96 was prepared,which make an foundation for further investigations of the protein.