Fusion protein of ATPase domain of Hsc70 with TRP2 acting as a tumor vaccine against B16 melanoma.
HSP70s are a family of ATP-dependent chaperones of relative molecular masses around 70kDa. Immunization of mice with HSP70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor. Recent researches have demonstrated that the ATPase domain of HSP70 and the tumor antigenic peptide that binds to Hsp70 were the crucial parts eliciting tumor-specific immunity. These findings suggested that a recombinant protein expressed in Escherichia coli, comprising a covalently fused fragment of tumor rejection antigen to ATPase domain of HSP70, could be used as a tumor vaccine. However, high-level expressions of heterologous recombinant proteins in E. coli often lead to the formation of inclusion bodies, resulting in defects in solubility and bioactivity. In the present work, we found an approach to resolve these problems, focusing on a refolding procedure via gel-filtration chromatography for denatured inclusion body proteins. Here, we expressed, purified and refolded a fusion protein comprising murine heat shock cognate protein 70 (Hsc70) N-terminal ATPase domain (Hsc70NTD) and a portion of TRP2 (aa153-417) as a model protein. The refolding effectivities were assessed according to their ATPase activities, the vaccine function was assessed according to immunization effect in inducing antigen-specific CTLs and to in vivo tumor protection. The results showed that the fusion protein refolded via gel-filtration chromatography exhibited ATPase activity, succeeded in eliciting antigen-specific CTL in vivo and delayed tumor growth on tumor-bearing mice.
Department of Biochemistry, School of Life Sciences, Fudan University, 200433 Shanghai, PR China.
Honghai
H
Zhang
Weirong
W
Wang
Qiujun
Q
Li
Weida
W
Huang
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000818:Animals; D019496:Cancer Vaccines / Q000276:immunology*; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D018920:Coculture Techniques; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D007694:Killer Cells, Natural / Q000187:drug effects; D008297:Male; D008545:Melanoma / Q000276:immunology* / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation / Q000473:pathology; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000009:adverse effects / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D019496:Cancer Vaccines; D050883:HSC70 Heat-Shock Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C113139:Trp2 protein, vertebrate; D000251:Adenosine Triphosphatases