Purification and partial characterization of outer membrane proteins P5 and P6 from Haemophilus influenzae type b

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Abstract
The major outer membrane proteins of H. influenzae type b (Hib), designated P5 and P6 were purified to homogeneity and partially characterized. P5 was insoluble in octylglucoside-NaCl and could be extracted with 1% sodium dodecyl sulfate (SDS) in 20 mM phosphate (pH 7.5). Solubilized P5 was further purified on hydroxylapatite in 0.1% SDS. The purified protein had an apparent MW of 27,000 as determined by SDS-polyacrylamide gel electrophoresis after sample preparation at room temperature. The protein migrated with an apparent MW of 35,000 after heating for 30 min at 100.degree. C in the presence of 10% .beta.-mercaptoethanol (.beta.ME). Rabbit antisera prepared against the purified preparation immunoprecipitated solubilized protein P5 but had no protective activity in the infant rat bacteremic model. The SDS-insoluble residue was further extracted with 1% SDS-0.5 M NaCl-0.1% .beta.ME at 37.degree. C. A single outer membrane protein, designated P6, with an apparent MW of 16,000, remained insoluble under these conditions. Antiserum prepared against this insoluble fraction contained antibodies which, after removal of anti-lipopolysaccharide antibody, immunoprecipitated P6 and protected infant rats challenged with Hib. Protein P6 could be released from the insoluble cell wall in the presence of SDS-NaCl-.beta.ME at 60.degree. C. Thus,proteins P5 and P6 could be purified from the cell envelope of Hib. Based on the results from infant rat passive protection experiments, antigens in the P6-cell wall fraction merit further investigation as possible vaccine components. Epitopes on protein P5 did not appear to elicit protective antibody.