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A hybrid outer membrane protein antigen for vaccination against Pseudomonas aeruginosa
J Gabelsberger1, B Knapp, S Bauersachs
1Institut für Biochemie, Ludwig-Maximilians-Universität München, Germany.
Abstract:
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.
Insights
Researchers developed a new method to express a hybrid protein from Pseudomonas aeruginosa in E. coli, aiming to create a vaccine against P. aeruginosa infections without using glutathione-S-transferase.
Area of Science:
- Microbiology
- Protein Engineering
- Vaccine Development
Background:
- Pseudomonas aeruginosa is a significant opportunistic pathogen.
- Outer membrane proteins OprF and OprI are targets for P. aeruginosa vaccines.
- Previous attempts used glutathione-S-transferase (GST), necessitating its removal.
Purpose of the Study:
- To express a hybrid OprF-OprI protein in E. coli without GST.
- To optimize the expression and purification of the hybrid protein.
- To prepare the hybrid protein for testing its protective efficacy against P. aeruginosa.
Main Methods:
- Constructing E. coli strains expressing the OprF-OprI hybrid protein with various modifications.
- Utilizing signal peptides to enhance periplasmic expression.
- Employing N-terminal extensions and histidine tags (N-terminal and C-terminal) for improved cytoplasmic expression and purification.
- Purifying the hybrid protein using immobilized-metal affinity chromatography (IMAC) under native and denaturing conditions.
Main Results:
- Signal peptides increased hybrid protein yield to 30% of total cell protein, but processing and periplasmic isolation were limited.
- An N-terminal extension of 11 amino acids from OprF significantly enhanced cytoplasmic expression.
- Attaching a six-histidine tag to the N-terminus further increased expression levels.
- Purification of N-terminal extended and N-terminal his-tagged OprF-OprI was successful via IMAC.
Conclusions:
- Optimized expression strategies in E. coli enable high-yield production of the OprF-OprI hybrid protein.
- The developed purification methods allow for the isolation of the hybrid protein for further immunological evaluation.
- These findings pave the way for developing a GST-free P. aeruginosa vaccine candidate.