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Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B
Fakhri Haghi1, Shahin Najar Peerayeh, Seyed Davar Siadat
1Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran. fakhri.haghi@gmail.com.
Introduction:
Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA.
Methodology:
A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porA plasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification.
Results:
Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle.
Conclusion:
This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.
Insights
This study successfully cloned and expressed the PorA protein from Neisseria meningitidis serogroup B using a prokaryotic system. This recombinant PorA protein is a potential candidate for developing vaccines against bacterial meningitis.
Area of Science:
- Microbiology
- Vaccinology
- Molecular Biology
Background:
- Neisseria meningitidis serogroup B causes meningitis and septicemia, with no current vaccines available.
- PorA, a major outer membrane protein of N. meningitidis, functions as a cationic porin.
- Developing vaccines against N. meningitidis serogroup B is a critical public health need.
Purpose of the Study:
- To clone the porA gene from N. meningitidis serogroup B.
- To establish a prokaryotic expression system for producing recombinant PorA protein.
- To verify the expression and characteristics of the recombinant PorA.
Main Methods:
- PCR amplification and cloning of the 1200 bp porA gene into the pET-32a vector.
- Transformation into Origami B (DE3) cells and induction with IPTG for protein overexpression.
- Purification using Ni-NTA agarose, followed by SDS-PAGE and Western blotting for analysis.
Main Results:
- Successful cloning of porA confirmed by PCR and enzymatic digestion, with 97% nucleotide sequence homology.
- Efficient induction of recombinant PorA protein (65 kDa) using 1.0 mmol/L IPTG.
- The recombinant PorA was expressed as inclusion bodies and showed reactivity with antiserum against native outer membrane vesicles.
Conclusions:
- A robust prokaryotic expression system for recombinant PorA production was established.
- This system offers a straightforward method for generating PorA for potential vaccine development.
- The approach may be applicable to producing other bacterial outer membrane proteins for vaccine research.
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