Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis

FEBS Letters

Srikumar, Ramakrishnan²  Coulton, James W.²  Laprade, Raynald¹  Dahan, David² 

[1] Groupe de Recherche en Transport Membranaire, Université de Montréal, Montreal H3C 3J7, Canada;Department of Microbiology and Immunology, McGill University, Montreal H3A 2B4, Canada

关键词: Porin;    Membrane channel;    Inclusion body;    Refolding;    Haemophilus influenzae type b;    Hib;    Haemophilus influenzae type b;    mAb;    monoclonal antibody;    LPS;    lipopolysaccharide;    CD;    circular dichroism;    FPLC;    fast protein liquid chromatography;    PAGE;    polyacrylamide gel electrophoresis;    ELISA;    enzyme-linked immunosorbent assay;    PBS;    phosphate-buffered saline;   

DOI  :  10.1016/0014-5793(96)00841-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The major diffusion channel in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; M _r 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.

【 授权许可】

Unknown