High expression of HCV core protein: a comparative study on effect of 6xHis-tag location (N- versus C-terminal) and purification method for various properties
Abstract number: 1733_1377
Sadat M., Aghasadeghi M., Bahramali G., Rouhvand F.
Objectives: Hepatitis C virus (HCV) core protein (HCVcp) as a multifunctional and conserved protein among various HCV genotypes is important for diagnosis, vaccine formulation experiments and studies on HCVcp-mediated pathogenesis. For such studies high amounts of pure HCVcp might be prepared by heterologous expression. Insertion of His-tag for purification of recombinant proteins has found great application in different laboratories; however, this procedure may have unwanted effects on conformation and antigenic properties. In this study, characterisation of a new E. coli-derived HCVcp as N- or C-terminally His-tagged protein (N- or C-HCVcp), purified in both native and denatured condition is provided.
Methods: The hydrophilic section of HCVcp gene (aa 2122) was inserted into pIVEX 2.3 and 2.4a which provided a 6xHis-tag at C- or N-terminal of the protein, respectively. E. coli BL21-AI harbouring constructs were induced by addition of L-Arabinose. HCVcp was purified in both native and denatured condition by NI-NTA agarose and characterised by SDS-PAGE, Immunoblotting and SELDI-TOF mass spectrometry. Antigenic and immunogenic properties of HCVcp were evaluated with HCV-infected human and immunised mice sera by ELISA respectively. Ability of particulate formation of proteins was examined by immuno-gold electron microscopy.
Results: The yields of protein expression were 25 and 16 mg/L in denatured versus 7 and 4 mg/L in native purification for N- versus C-HCVcp, respectively. N-terminal fragmented products of 9 and 11 Kd, which were not due to proteolytic activity but apparently result of ribosomal release were identified. However, these fragmented products were not purified with C-HCVcp. Diagnostic properties of natively purified proteins were predominant and still better for C-HCVcp, However N-HCVcp reacted with C-HCVcp-Immunised mice sera in lower titers. Only natively purified proteins were capable of particulate formation and assembling to generate VLPs.
Conclusion: Purification in denatured/refolding condition may not result to proper conformation of HCVcp, thereby native purification may be undertaken for any kind of applications. C-HCVcp which can be purified as a homogenous product is predominant for diagnostic and pathogenic studies while N-HCVcp that is purified as both fragmented and complete products may be used for generation of antibodies because of better presentation of linear epitopes which are mostly located on the N-terminal of HCVcp.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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