title: Expression of large amounts of neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers.
abstract: Porins from different neisserial strains and species have been shown to have differences in both primary amino acid sequence and biophysical characteristics as observed by functional assays. A closer examination of how the changes in the primary amino acid sequence of Neisseria porin molecules correlate with these observed biophysical changes has been impeded by the inability to easily manipulate the cloned porin genes by modern molecular techniques and then obtain enough of the expressed modified porin protein to purify and use in these biophysical functional assays. In this report, we describe a method by which the genes encoding three different porin proteins, lacking their neisserial promoter and signal sequences, were cloned into an expression plasmid and transformed into Escherichia coli. Upon induction, large amounts of the porin proteins were produced. The expressed porin proteins were then manipulated to regenerate their native trimer structure and purified by standard protein chemistry. Sufficient purified recombinant porin protein was obtained for further antigenic as well as biophysical characterization. This sets the stage for the biophysical characterization of these neisserial porin proteins in more detail.
authors.affiliation: Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University, New York 10021.
authors.forename: H L
authors.initials: HL
authors.lastname: Qi
authors.forename: J Y
authors.initials: JY
authors.lastname: Tai
authors.forename: M S
authors.initials: MS
authors.lastname: Blake
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009343:Neisseria / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D018272:Porins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D018272:Porins; D011994:Recombinant Proteins