title: [Cloning, expression and purification of the C-terminal section of murine heat shock protein gp96].
abstract: Heat shock protein gp96 is a glycoprotein which was found several years ago. Besides its function as a molecular chaperone, it is also reported to play important roles both in innate immunity and adaptive immunity. Gp96 can stimulate the maturation of antigen presenting cells (especially dendritic cells) and the secretion of cytokines. Gp96 and its associated peptides could stimulate peptide specific cytotoxic T lymphocyte reaction (CTL), which was very promising in the designing of anti-virus and anti-tumor vaccines. However the expression level of whole length gp96 was relatively low in E. coli and the purity of gp96 are not very suitable for further study. We successfully cloned the carboxy terminal fragment (560aa-751aa) of murine gp96 into the pGEX-6p-1 vector and expressed in BL21 strain. This fragment contains the peptide binding domain and the dimerization domain. After purification, the recombinant fusion protein was cleaved with the PreScission Protease and analyzed by Gelfiltration. The results show that this fragment may be related to the dimerization of gp96 and make an foundation for further investigations of the protein.
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Jin-Le
authors.initials: JL
authors.lastname: Han
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Li
authors.forename: Ji-Lin
authors.initials: JL
authors.lastname: Li
authors.forename: Po
authors.initials: P
authors.lastname: Tien
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics*; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000951:Antigens, Neoplasm; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C052646:sarcoma glycoprotein gp96 rejection antigens