Studies On Prokaryotic Expression And Structural Prediction Of The Quorum-sensing-related Protein CqsA Of Vibrio Parahaemolyticus

[Objective] Quorum sensing, a population-density-related genetic regulatory mechanism, depends on the production, secretion, accumulation, and detection of diffusible signaling molecules called Autoinducers. CqsA, a quorum-sensing-related protein in Vibrio parahaemolyticus, is a putative autoinducer synthase, and the autoinducer synthesized by CqsA maybe is a major regulator in quorum sensing system. [Methods] In this paper, the gene cqsA was cloned from V. parahaemolyticus ATCC17802 and inserted into the expression vector pET22b. After sequencing the recombinant vector pET22b-cqsA was transformed into Escherichia coli BL21 and induced by IPTG, then the fusion protein was analyzed by SDS-PAGE and verified by Western Blotting. After sequences alignment between V. parahaemolyticus ATCC17802 CqsA (CqsAATCC17802) and V. cholera N16961 CqsA (CqsAN16961), the protein CqsAATCC17802 was modeled and analyzed by using the software Discovery Studio 2.1. [Results] SDS-PAGE results showed that the pET22b-cqsA could be able to express efficiently in a form of inclusion body under the induction of 0.6 mM IPTG for 4h. The molecular weight of the fusion protein was 49kD, and this result matched the theory value (437aa). The sequence identity and similarity between CqsAATCC17802 and CqsAN16961 shared 57.6% and 77.9% levels, respectively. Homology Modeling results indicated that V. parahaemolyticus CqsA was a pyridoxal-5'-phosphate (PLP)-dependent enzyme with acyltransferase activity, and its monomer had a PLP binding site in the cavity composed by Trp113, Asp177, Asp206, His209 and Lys240. [Conclusion] In this experiment, we succeed in expressing the gene cqsA in E. coli, and analysed the structure of CqsAATCC17802.We predicted that the CqsAs of V. parahaemolyticus and V. cholerae had similar function, and CqsAATCC17802 might synthesize the similar autoinducer as CqsAN16961.Our study laid solid foundation for follow-up tracing of signaling molecules synthesized by CqsAATCC17802 exploration of quorum-sensing system in V. parahaemolyticus.