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Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis
Author(s) -
Dahan David,
Srikumar Ramakrishnan,
Laprade Raynald,
Coulton James W.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00841-1
Subject(s) - porin , bacillus subtilis , recombinant dna , microbiology and biotechnology , haemophilus influenzae , chemistry , haemophilus , escherichia coli , bacteria , biology , biochemistry , bacterial outer membrane , genetics , antibiotics , gene
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.