Expression of large amounts of neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers.
AUTOR(ES)
Qi, H L
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=186529Documentos Relacionados
- Location and distribution of the light chains in clathrin trimers.
- Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12.
- Functional Expression in Escherichia coli and Membrane Topology of Porin HopE, a Member of a Large Family of Conserved Proteins in Helicobacter pylori
- Domains required for assembly of adenovirus type 2 fiber trimers.
- Expression and assembly of active cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits.