title: Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70.
abstract: The aim of this study was to express and purify recombinant proteins based on human prostate stem cell antigen (PSCA) and heat shock protein-70 (HSP70). The PSCA gene and various structural domains of HSP70 were amplified by polymerase chain reaction (PCR) with the respective primers. Then, the PSCA was cloned into the prokaryotic expression vector pET21a(+) with the amino-terminus, carboxyl-terminus and overall length of HSP70, by enzyme digestion to construct the recombinant plasmids pET21-PSCA-HSPN, pET21-PSCA-HSPC and pET21-PSCA-HSP, respectively. After being expressed inEscherichia coli (E. coli)by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, recombinant fusion proteins were purified. Western blotting was performed to confirm the expression of the recombinant proteins. The results revealed that recombinant plasmids were successfully constructed. The PSCA-HSPC and PSCA-HSP expressed inE. coliexisted in soluble form, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the recombinant proteins PSCA-HSPC and PSCA-HSP reached >95% following purification with the nickel-nitrilotriacetic acid (Ni-NTA) resin, Phenyl-Sepharose Fast Flow and Superdex 75, which lays a foundation for the development of vaccines for prostate cancer.
authors.affiliation: Clinical Laboratory Center, PLA Air Force General Hospital, Haidian, Beijing 100142, P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Dong
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Zhang
authors.forename: Changming
authors.initials: C
authors.lastname: Yu
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
authors.forename: Lihua
authors.initials: L
authors.lastname: Hou
authors.forename: Ling
authors.initials: L
authors.lastname: Fu
authors.forename: Shaoqiong
authors.initials: S
authors.lastname: Yi
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
keywords: heat shock protein; prostate stem cell antigen; recombinant fusion protein; structural domain