Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70

Dong,Lei; Zhang,Xiaopeng; Yu,Changming; Ren,Jun; Hou,Lihua; Fu,Ling; Yi,Shaoqiong; Chen,Wei

doi:10.3892/etm.2013.967

Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70

Authors:
- Lei Dong
- Xiaopeng Zhang
- Changming Yu
- Jun Ren
- Lihua Hou
- Ling Fu
- Shaoqiong Yi
- Wei Chen
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Affiliations: Clinical Laboratory Center, PLA Air Force General Hospital, Haidian, Beijing 100142, P.R. China, Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Fengtai, Beijing 100071, P.R. China
Published online on: February 20, 2013 https://doi.org/10.3892/etm.2013.967
Pages: 1161-1164

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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 in Escherichia 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 in E. coli existed 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.

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1	Reiter RE, Gu Z, Watabe T, et al: Prostate stem cell antigen: A cell surface maker overexpressed in prostate cancer. Proc Natl Acad Sci USA. 95:1735–1740. 1998. View Article : Google Scholar : PubMed/NCBI
2	Gu Z, Thomas G, Yamashiro J, et al: Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer. Oncogene. 19:1288–1296. 2000. View Article : Google Scholar : PubMed/NCBI
3	Krupa M, Canamero M, Gomez CE, Najera JL, Gil J and Esteban M: Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression. Vaccine. 29:1504–1513. 2011. View Article : Google Scholar : PubMed/NCBI
4	Garcia-Hernandez Mde L, Gray A, Hubby B, Klinger OJ and Kast WM: Prostate stem cell antigen vaccination induces a long-term protective immune response against prostate cancer in the absence of autoimmunity. Cancer Res. 68:861–869. 2008.PubMed/NCBI
5	Ahmad S, Casey G, Cronin M, Rajendran S, Sweeney P, Tangney M and O’Sullivan GC: Induction of effective antitumor response after mucosal bacterial vector mediated DNA vaccination with endogenous prostate cancer specific antigen. J Urol. 186:687–693. 2011. View Article : Google Scholar
6	Srivastava PK and Udono H: Heat shock protein-peptide complexes in cancer immunotherapy. Curr Opin Immunol. 6:728–732. 1994. View Article : Google Scholar : PubMed/NCBI
7	Srivastava PK, Udono H, Blachere NE and Li Z: Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics. 39:93–98. 1994. View Article : Google Scholar : PubMed/NCBI
8	Blachere NE and Srivastava PK: Heat shock protein-based cancer vaccines and related thoughts on immunogenicity of human tumors. Semin Cancer Biol. 6:349–355. 1995. View Article : Google Scholar : PubMed/NCBI
9	Matsueda S, Kobayashi K, Nonaka Y, Noguchi M, Itoh K and Harada M: Identification of new prostate stem cell antigen-derived peptides immunogenic in HLA-A2(+) patients with hormone-refractory prostate cancer. Cancer Immunol Immunother. 53:479–489. 2004.PubMed/NCBI
10	Bahrenberg G, Brauers A, Joost HG and Jakse G: PSCA expression is regulated by phorbol ester and cell adhesion in the bladder carcinoma cell line RT112. Cancer Lett. 168:37–43. 2001. View Article : Google Scholar : PubMed/NCBI
11	Argani P, Rosty C, Reiter RE, et al: Discovery of new markers of cancer through serial analysis of gene expression: prostate stem cell antigen is overexpressed in pancreatic adenocarcinoma. Cancer Res. 61:4320–4324. 2001.PubMed/NCBI
12	Dannull J, Diener PA, Prikler L, et al: Prostate stem cell antigen is a promising candidate for immunotherapy of advanced prostate cancer. Cancer Res. 60:5522–5528. 2000.PubMed/NCBI
13	Saffran DC, Raitano AB, Hubert RS, Witte ON, Reiter RE and Jakobovits A: Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts. Proc Natl Acad Sci USA. 98:2658–2663. 2001. View Article : Google Scholar : PubMed/NCBI
14	Blachere NE, Li Z, Chandawarkar RY, et al: Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med. 186:1315–1322. 1997. View Article : Google Scholar
15	Srivastava PK: Immunotherapy for human cancer using heat shock protein-peptide complexes. Curr Oncol Rep. 7:104–108. 2005. View Article : Google Scholar : PubMed/NCBI
16	Chu NR, Wu HB, Wu T, Boux LJ, Siegel MI and Mizzen LA: Immunotherapy of a human papillomavirus (HPV) type 16 E7-expressing tumour by administration of fusion protein comprising Mycobacterium bovis bacille Calmette-Guérin (BCG) hsp65 and HPV16 E7. Clin Exp Immunol. 121:216–225. 2000. View Article : Google Scholar : PubMed/NCBI
17	Liu DW, Tsao YP, Kung JT, Ding YA, Sytwu HK, Xiao X and Chen SL: Recombinant adeno-asseciated virus expressing human papilomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer. J Virol. 74:2888–2894. 2000. View Article : Google Scholar
18	Zhang X, Yu C, Zhao J, et al: Vaccination with a DNA vaccine based on human PSCA and HSP70 adjuvant enhances the antigen-specific CD8⁺ T-cell response and inhibits the PSCA⁺ tumors growth in mice. J Gene Med. 9:715–726. 2007. View Article : Google Scholar : PubMed/NCBI
19	Zhu X, Zhao X, Burkholder WF, Gragerov A, Ogata CM, Gottesman ME and Hendrickson WA: Structural analysis of substrate binding by the molecular chaperone DnaK. Science. 272:1606–1614. 1996. View Article : Google Scholar : PubMed/NCBI
20	Wang Y, Kelly CG, Singh M, McGowan EG, Carrara AS, Bergmeier LA and Lehner T: Stimulation of TH1-polarizing cytokines, C-C chemokines, maturation of dendritic cells and adjuvant function by the peptide binding fragment of heat shock protein 70. J Immunol. 169:2422–2429. 2002. View Article : Google Scholar