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. 1990 Dec;58(12):3833–3839. doi: 10.1128/iai.58.12.3833-3839.1990

Purification and characterization of an outer membrane protein adhesin from Haemophilus parainfluenzae HP-28.

C H Lai 1, C Bloomquist 1, W F Liljemark 1
PMCID: PMC313743  PMID: 2254013

Abstract

Outer membranes were isolated from Haemophilus parainfluenzae HP-28 by a mild extraction method followed by Sephadex G-150 gel filtration chromatography. The first peak (pool 1) recovered contained an activity which inhibited adherence of HP-28 cells to saliva-coated spheroidal hydroxyapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pool 1 revealed a dominant protein band of 34 kDa. The SDS-PAGE-purified 34-kDa protein was excised from the gel and used for antibody preparation in rabbits. The antiserum produced was analyzed by immunoblot and was shown to be monospecific for the 34-kDa protein. Anti-34-kDa protein antibody was purified from the rabbit antiserum by protein A-Sepharose 6MB affinity chromatography. This antibody was then cross-linked to protein A-Sepharose 6MB to construct a second affinity column. The 34-kDa proteins were purified from outer membranes by this affinity chromatography. The 34-kDa protein was homogeneous, as confirmed by SDS-PAGE, isoelectric focusing, and reverse-phase chromatography analyses. Fab and Fc fragments of the purified anti-34-kDa protein antibodies were prepared by papain digestion, followed by carboxymethyl cellulose chromatography. Fab fragments from the anti-34-kDa protein antibody and the affinity-purified 34-kDa protein both showed significant inhibition of parent H. parainfluenzae HP-28 cell adherence to experimental salivary pellicle and to Streptococcus sanguis SA-1.

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Selected References

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