title: Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran. fakhri.haghi@gmail.com.
authors.forename: Fakhri
authors.initials: F
authors.lastname: Haghi
authors.forename: Shahin Najar
authors.initials: SN
authors.lastname: Peerayeh
authors.forename: Seyed Davar
authors.initials: SD
authors.lastname: Siadat
authors.forename: Mehran
authors.initials: M
authors.lastname: Montajabiniat
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D008970:Molecular Weight; D038541:Neisseria meningitidis, Serogroup B / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D018272:Porins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D018272:Porins; D011994:Recombinant Proteins; C057699:porin protein, Neisseria