title: Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.
abstract: In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.
authors.affiliation: Meningococcal Research Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: Daniel
authors.initials: D
authors.lastname: Yero
authors.forename: Rolando
authors.initials: R
authors.lastname: Pajón
authors.forename: Olivia
authors.initials: O
authors.lastname: Niebla
authors.forename: Gretel
authors.initials: G
authors.lastname: Sardiñas
authors.forename: Isbel
authors.initials: I
authors.lastname: Vivar
authors.forename: Yasser
authors.initials: Y
authors.lastname: Perera
authors.forename: Darien
authors.initials: D
authors.lastname: García
authors.forename: Maité
authors.initials: M
authors.lastname: Delgado
authors.forename: Karem
authors.initials: K
authors.lastname: Cobas
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins