A hybrid outer membrane protein antigen for vaccination against Pseudomonas aeruginosa.
Recently a hybrid protein containing parts of the outer membrane proteins OprF (aa 190-342) and OprI (aa 21-83) from Pseudomonas aeruginosa fused to the glutathione-S-transferase was shown to protect mice against a 975-fold 50% lethal dose of P. aeruginosa. To omit the use of the GST-protein, the hybrid protein OprF-OprI was expressed in E. coli using distinct modifications which have not to be eliminated after its expression. Using different signal peptides, the yield of the hybrid protein OprF-OprI in E. coli could be increased to 30% of the total cell protein, however, only a very small amount of the hybrid preprotein was processed and could be isolated from the periplasm of the host. A construct containing an N-terminal extension of 11 amino acids from the original OprF gene gave rise to a significantly higher expression in the cytoplasm. Purification was facilitated by the addition of a five histidine tag at the C-terminus. An even higher expression was obtained by a construct in which a six histidine tag was attached to the N-terminus of the hybrid protein. The N-terminal extended OprF-OprI as well as the N-terminal his-tagged OprF-OprI hybrid antigens were purified by immobilized-metal affinity chromatography under native and denaturing conditions and can now be tested for protectivity against P. aeruginosa in animal model systems.
Gabelsberger
J
J
Institut für Biochemie, Ludwig-Maximilians-Universität München, Germany.
Knapp
B
B
Bauersachs
S
S
Enz
U I
UI
von Specht
B U
BU
Domdey
H
H
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D001428:Bacterial Vaccines; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D006639:Histidine; D008074:Lipoproteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018272:Porins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D016324:Sequence Tagged Sites; D014614:Vaccines, Synthetic
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D017931:DNA Primers; D008074:Lipoproteins; D018272:Porins; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C060507:outer membrane lipoprotein I, Bacteria; D006639:Histidine; D005982:Glutathione Transferase