Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis.
The major diffusion channel in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; Mr 37 782). The Hib porin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti-Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.
Dahan
D
D
Department of Microbiology and Immunology, McGill University, Montreal, Canada.
Srikumar
R
R
Laprade
R
R
Coulton
J W
JW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D003902:Detergents; D006193:Haemophilus influenzae / Q000737:chemistry* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D018272:Porins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003902:Detergents; D008070:Lipopolysaccharides; D018272:Porins; D011994:Recombinant Proteins; D014357:Trypsin