Purification and characterization of an outer membrane protein adhesin from Haemophilus parainfluenzae HP-28.
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.
Lai
C H
CH
Department of Diagnostic School of Dentistry, University of Minnesota, Minneapolis 55455.
Bloomquist
C
C
Liljemark
W F
WF
D000818:Animals; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D044622:Dental Pellicle; D004591:Electrophoresis, Polyacrylamide Gel; D006190:Haemophilus / Q000032:analysis*; D007525:Isoelectric Focusing; D011817:Rabbits
D001425:Bacterial Outer Membrane Proteins