Purification and renaturation of membrane neuraminidase from Haemophilus parasuis.
Haemophilus parasuis, which causes polyserositis, polysynovitis, meningitis, septicemia, and pneumonia in pigs, has emerged as an increasing problem in modern swine production systems. Co-factors for and the pathogenesis of H. parasuis disease are not defined. One of the potential virulence factors of H. parasuis is its neuraminidase (sialidase). While purifying the H. parasuis neuraminidase from the membrane fraction, we developed a protocol to renature enzymatic activity after enzyme preparations were resolved electrophorectically in denaturing polyacrylamide gels. The H. parasuis neuraminidase co-resolved with recombinant neuraminidase of Vibrio cholera; thus its apparent molecular mass is 82 kilodalton (kDa). The H. parasuis neuraminidase was associated with the membrane fraction and the purification protocol removed over 99% of the H. parasuis cell protein while retaining over 90% of the neuraminidase activity. Purified protein will provide another avenue to clone the neuraminidase gene that has been refractory to cloning and the protocol will be a means to purify recombinant protein.
Lichtensteiger
Carol A
CA
Department of Pathobiology, Veterinary Diagnostic Laboratory, University of Illinois Urbana-Champaign Campus, 2001 South Lincoln Avenue, Urbana, IL 61802, USA. clichten@uiuc.edu
Vimr
Eric R
ER
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D006190:Haemophilus / Q000201:enzymology*; D008970:Molecular Weight; D009439:Neuraminidase / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation / Q000502:physiology; D020673:Protein Renaturation; D013552:Swine
D001425:Bacterial Outer Membrane Proteins; D009439:Neuraminidase