Structure Research On Pseudomonas Aeruginosa RNA Degradation Protein PNPASE And Flagella Cap Protein FLID

Background The RNA degradosome is the key tool of RNA turnover. RraA is the improtant inhibiting factor in the process of RNA degradation by RNA degradosome. Flagella were the improtant composite in the bacteria, and also is one of virulence factor in the bacteria. In our previous study, The structure of flagella protein FlgD have been determined and the RraA protein have been purified. We want to clarify the mechanism of the RNA degradation by the structure study of PNPase and RraA, and understand the assemble mechanism of flagella via the structure study of FliD.Objectives The purpose of this study was to purify the PNPase, RraA and FliD protein, and obtain protein crystal, then collect X-ray X-Ray diffraction data of the protein crystal. Use this data to analyse three-dimensional structure of these protein.Methods Bioinformatics analysis of these protein were viaed the internet. Using the molecular cloning technique, we cloned, expressed and purified the high concentration and purity protein. State of aggregation was attained through the analysis with molecular sieve chromatography column Superdex 200. The high quality protein crystals were obtained by using commercialized reagent and optimizing the crystal conditions. Using the X-ray diffraction instrument collect crystal data, and using computer software analysis three-dimensional structure of protein. The PNPase-RNase E compound was obtained by the Pull-down technique.Results We succeeded in obtaining the high concentration and purity pseudomonas aeruginosa protein PNPase, RraA, RNase E and the compound of PNPase-RNase E, and Enteritis salmonella flagella cap protein FliD. Then we succeeded in cultivating the crystal of PNPase, RraA, FliD, and collecting data of PNPase and RraA. The molecular sieve test showed PNPase,RraA and FliD were peNATmer,Dimer of trimer,and Dimer of Dimer respectively.