Cloning, Expression and Purification of the Factor H Binding Protein and Its Interaction With Factor H



Yarian F¹ ; Bandehpour M^{2, 3} ; Seyed N⁴ ; Kazemi B^{1, 2, 3} Show Affiliations
Source: Iranian Journal of Microbiology Published:2016

Abstract

Background and Objective:Neisseria meningitidisis a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor ofNeisseria meningitidisthat is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogene- sis and vaccine design. The aims of present research were cloning, expression, purification of fHbp and confirmation of the interaction between serum factor H (fH) and produced factor H binding protein. Materials and Methods:A 820 base pairsfhbp gene fragment was amplified by PCR and cloned into expression vector pE- T28a (+) inBam HIandSalIrestriction enzymes sites. Recombinant DNA was expressed in BL21 (DE3) cell. fHBP protein was purified by Ni-NTA agarose resin. Coupling of recombinant protein into CNBr activated Sepharose 4B resin was carried out for application in serum fH protein purification. (fH-fHBP) interaction was confirmed by SDS-PAGE and far-western blotting. Results and Conclusions:SDS-PAGE results showed a 35 kDa protein band. 150 kDa fH protein was purified by designed Sepharose 4B resin. Far-western blotting confirmed (fH-fHBP) interaction and proper folding of factor H binding protein. © 2016, Tehran University of Medical Science. All Rights reserved.