Pseudomonas aeruginosa is a major cause of hospital-acquired infections, particularly in mechanically ventilated patients. it is also a leading cause of death in cystic fibrosis patients. a key virulence factor associated with the disease severity of p. aeruginosa is its type iii secretion system (t3ss) which injects bacterial toxins directly into the cytoplasm of host cells. pcrv is an important structural protein of the ttss. the desired anti-pcrv monoclonal antibody has been engineered.
in the current investigation, a recombinant scfv monoclonal antibody was developed against the pcrv protein and expressed in enbase (fed-batch) cultivation system.
Methods
The petitetm n-his sumo kan vector including pcrv gene was transformed into recombinant e.coli (bl21) cells which cultured in the enbase medium. the expression of recombinant protein was then induced by iptg. the expression and solubility of pcrv protein were investigated in two different temperatures (25 and 30°c) and different induction time (4, 6, 8, 12, 24 hours). protein expression was analyzed by sds-page and western blotting procedure
Results
Our results showed that enbase had higher efficiency than lb broth; the maximum level of solubility and total protein expression was observed in enbase (fed-batch) cultivation system after iptg induction.
Conclusion
Therefore, due to the slow and continuous release of glucose, a greater expression and solubility of pcrv-scfv protein can be achieved in fed-batch cultivation mode than common method of cultivation. also, sumo tag is an effective strategy to improve protein solubility.
