Study Of Expression,Purification And Function For NalD And CzcR,DNA Regulator For Drug Efflux Pumps Of Pseudomonas Aeruginosa

In Pseudomonas aeruginosa,there are several kinds of highly efficient efflux pump systems to help P.aeruginosa surviving in harsh environments by providing such as antibiotic resistance and heavy metal resistance.In P.aeruginosa the expressions of these efflux pumps genes are regulated by transcription regulators.It can help us to disclose more details about the molecular mechanism of drug resistance in P.aeruginosa by studing structures and functions of these regulators.NalD protein,one of regulators of multi-drug efflux pump MexAB-OprM in P.aeruginosa,was expressed and purified.It was occasionally found that NalD and PEG3350 had an unreported special interaction in solution,when the crystallization conditions for NalD protein were screened.In order to figure out the interaction between NalD and PEG3350,we used SDS-PAGE,MALDI-TOF,dynamic laser light scattering and analytical ultracentrifugation to study this phenomenon.It was found that NalD and PEG3350 formed a kind of complex by hydrogen bonds between the side chain groups in NalD and the ether oxygen groups in PEG3350,which had larger apparent molecular weight and smaller hydrodynamic size.In addition,CzcR,a regulator protein which involved in heavy metal ions resistance and imipenem resistance in P.aeruginosa,was also expressed and purified.And RT-PCR was used to figure out the influence as PAO1 treated by different concentrations of 2Zn2+.It was found that as the Zn2+ concentration increased in environment,both of the resistances to Zn2+ and imipenem increased.