ABSTRACT
The human papillomavirus L1 major capsid protein [HPV L1], the basis of the current vaccines, self-assembles into virus-like particles [VLPs]. Herein, we describe the expression and purification of recombinant HPV16 L1 in E. coli system. The L1 protein was generated in a fused form using an inducible expression system. The recombinant GST-L1 fusion protein migrated as a 82 kDa protein in SDS-PAGE. The L1 proteins formed inclusion bodies which were purified by Zn+2 reverse staining of sodium dodecyl sulfate polyacrylamide gels [SDS-PAGE] as a sensitive detection method. In western blotting, the existence of a 82 kDa band for GST-L1 protein was confirmed by anti-HPV16 L1 monoclonal antibody Camvir 1. The purified protein fraction was concentrated by ultrafiltration and dialyzed against PBS. This study has implications for the development of L1 protein purification as well as chromatographic separation used by other studies. Indeed, we could present a simple method to purify L1 protein in E. coli
ABSTRACT
The development of a therapeutic vaccine against human papillomavirus [HPV] is important for the control of cervical cancer. E7 is the major transforming protein produced in cervical cancers, and therefore represents potential tumor-specific antigen that could be the target of immunotherapy for cervical cancer. Among different vaccine strategies, protein-based vaccines are capable of generating CD8+ T cell responses in vaccinated animals and humans. Recently, development of novel strategies that enhance protein vaccine potency is important for generation of effective cancer vaccines and immunotherapies. Heat shock proteins [HSPs] including Gp96 have been shown to act as potent immuno-adjuvant to enhance antigen-specific tumor immunity. Therefore, the HSP-based protein vaccines can be administered by fusing antigens to HSPs, in vitro. It has been known that the HSP fragments [e.g., N-/or C-terminal regions] as mini-chaperones are better choice for immunization. The most straightforward method to produce large amounts of recombinant protein suitable for a vaccine is to clone the gene into a prokaryotic expression vector and produce the protein in Escherichia coli. In current study, we describe cloning of the HPV16 E7 gene linked to C-terminal fragment of gp96, identification and purification of the resultant E7-CT [gp96] fusion protein for next usage as a potential vaccine candidate protein against HPV in a pre-clinical trial. The recombinant E7-CT [gp96] migrated as a 51 kDa protein in SDS-PAGE. In Western blot experiment, the existence of a 51 kDa band for rE7-CT [gp96] was confirmed by rabbit anti-His as well as mouse anti-HPV16 E7 monoclonal antibodies. The protein of interest was both in the insoluble and the soluble fraction; therefore, purification was performed under denaturating and native conditions by affinity chromatography on Ni-NTA resin using 6xHis-tag