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, commonly associated with otitis media and exacerbations of chronic bronchitis. Studies concerning the pathogenesis of NTHi have proposed an important function for P5, an outer membrane protein believed to play a role in the initiation of infection by mediating adherence to respiratory mucin. P5 has also generated interest as a potential vaccine candidate. In a previous study, an NTHi library screen with antibodies raised against P5 purified from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified protein was contaminated with closely migrating proteins. Consequently, the aim of this study was to express P5 in a heterologous system to overcome potential contamination with NTHi proteins that may complicate analytical or vaccine studies. Recombinant P5, with an N terminal extension of 10 residues that included six histidines, was cloned and expressed in Escherichia coli. The rP5 was purified with the Talon metal affinity resin in a denatured form and then refolded by incorporation into mixed-detergent micelles of octylglucoside and SDS. Circular dichroism of the refolded rP5 demonstrated 55% beta-strand content, which is consistent with the beta-strand content of native P5 and the homologous E. coli protein OmpA.