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. 1985 Sep;49(3):544–549. doi: 10.1128/iai.49.3.544-549.1985

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

R S Munson Jr, D M Granoff
PMCID: PMC261198  PMID: 2411657

Abstract

The major outer membrane proteins of Haemophilus influenzae type b (Hib), designated P5 and P6 (R.S. Munson, Jr., J.L. Shenep, S.J. Barenkamp, and D.M. Granoff, J. Clin. Invest. 72:677-684, 1983), 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 molecular weight of 27,000 as determined by SDS-polyacrylamide gel electrophoresis after sample preparation at room temperature. The protein migrated with an apparent molecular weight of 35,000 after heating for 30 min at 100 degrees 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 degrees C. A single outer membrane protein, designated P6, with an apparent molecular weight 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 degrees 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. In contrast, epitopes on protein P5 did not appear to elicit protective antibody.

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Selected References

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