Cloning, expression and purification of the factor H binding protein and its interaction with factor H
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Published on: January 8, 2014
Fatemeh Yarian1, Mojgan Bandehpour2, Negar Seyed3
1Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Background And Objective:
Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor of Neisseria meningitidis that is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogenesis 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 pairs fhbp gene fragment was amplified by PCR and cloned into expression vector pET28a (+) in Bam HI and SalI restriction 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.
Insights
Researchers cloned and expressed the factor H binding protein (fHBP) from Neisseria meningitidis. They confirmed its interaction with human factor H, crucial for understanding meningitis pathogenesis and developing vaccines.
Area of Science:
- Molecular Biology
- Immunology
- Microbiology
Background:
- Neisseria meningitidis is a primary cause of meningitis and sepsis globally.
- Factor H binding protein (fHBP) is a critical Neisseria meningitidis virulence factor.
- fHBP binds human factor H, regulating the alternative complement pathway, impacting pathogenesis and vaccine development.
Purpose of the Study:
- To clone, express, and purify the fHBP from Neisseria meningitidis.
- To confirm the interaction between the produced fHBP and human factor H (fH).
Main Methods:
- Amplification and cloning of the fhbp gene fragment into the pET28a (+) vector.
- Expression of recombinant DNA in BL21 (DE3) cells and purification of fHBP using Ni-NTA agarose resin.
- Confirmation of fH-fHBP interaction via SDS-PAGE and far-western blotting after coupling fHBP to CNBr-activated Sepharose 4B.
Main Results:
- SDS-PAGE revealed a distinct 35 kDa protein band for the purified fHBP.
- A 150 kDa human factor H protein was successfully purified using the designed Sepharose 4B resin.
- Far-western blotting validated the interaction between fH and fHBP, indicating proper protein folding.
Conclusions:
- The study successfully cloned, expressed, and purified Neisseria meningitidis fHBP.
- The interaction between fHBP and human factor H was confirmed, validating the protein's functionality.
- These findings support the role of fHBP in meningococcal pathogenesis and its potential as a vaccine target.
