A method for the purification and refolding of a recombinant form of the nontypeable Haemophilus influenzae P5 outer membrane protein fused to polyhistidine

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen, com monly associated with otitis media and exacerbations of chronic bronchitis. Studies concerning the pathogenesis of NTHi have proposed an important fun ction for P5, an outer membrane protein believed to play a role in the init iation of infection by mediating adherence to respiratory mucin. P5 has als o generated interest as a potential vaccine candidate. In a previous study, an NTHi library screen with antibodies raised against P5 purified from sod ium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstra ted that the purified protein was contaminated with closely migrating prote ins. Consequently, the aim of this study was to express P5 in a heterologou s system to overcome potential contamination with NTHi proteins that may co mplicate analytical or vaccine studies. Recombinant P5, with an N terminal extension of 10 residues that included six histidines, was cloned and expre ssed in Escherichia coli. The rP5 was purified with the Talon metal affinit y resin in a denatured form and then refolded by incorporation into mixed-d etergent micelles of octylglucoside and SDS. Circular dichroism of the refo lded rP5 demonstrated 55% beta-strand content, which is consistent with the beta-strand content of native P5 and the homologous E. coli protein OmpA. (C) 1999 Academic Press.