The Expression And Purification Of Peptide Deformlyase And The Study Of Its Inhibitors

Objective:Peptide deformylase (PDF) is considered to be a promising target for antibacterial drug screening. In this study, we obtained the PDF of E.coli and S.auraus with high stability and activity and established the PDF inhibitors screening assay system. The effectiveness of PDF inhibitors were evaluated by determining the IC50 values, while determined the minimal inhibitory concentration (MIC) of PDF inhibitors to 8 bacteria. The work provided the basis for the further study and characterization of PDF and for the screening of new types of antibiotic drugs.Method:1. The expression plasmid pET-22b-def was transformed into E. coli BL21 (DE3) cells, then the cells were induced under different conditions to express PDF. After determination of the optimum expression conditions, we performed the multiple expression of E.coli PDF according to the above method.2. The E.coli PDF was purified by ion-exchange chromatography and size exclusion chromatography.3. The activity assays and stability assays of E.coli PDF were performed then the PDF inhibitors screening model was built up.4. The recombinant expression plasmid of S.aureus PDF was constructed.5. The expression plasmid was transformed into E.coli BL21 (DE3) cells, then the cells were induced and purified by agarose beads, obtained the PDF without ion, obtained the Ni2+-PDF by adding Nickel Chloride in the purification progress, and obtained the iron-enriched PDF by adding the Ferric Chloride in the expression and purification progress.6. The activity of three different metal forms S.aureus PDF were determined and compared, then the PDF inhibitors screening model was built up.7. The inhibition effect of PDF inhibitors on PDF was determined. 8. The MIC values of 39 PDF inhibitors to 4 Gram-positive bacteria and 4 Gram-negative bacteria were performed by microdilution susceptibility test.Resulte:1. The expression level of E.coli PDF was highest when E.coli BL21(DE3) cells were induced by 0.2mM for 4h under 30℃. We can get 30mg PDF from 1L E.coli BL21(DE3) cells.2. After ion-exchange chromatography and gel filtration chromatography, the hybrid proteins were removed, and highly purified PDF was obtained.3. The E.coli PDF with high activity and stability was obtained and applied to PDF inhibitors screening.4. Colony PCR and Gene sequencing confirmed that the expression plasmid of S.aureus PDF was constructed successfully.5. Three metal forms S.aureus PDF with high purity:PDF without metal ion, Ni2+-PDF and iron-enriched PDF were obtained under different expression and purification conditions.6. The activity of the iron-enriched PDF was the best among the three metal forms PDF, so it was used to establish the screening model.7. The inhibition effects of PDF inhibitors on PDF from E.coli and S.aureus were determined, but no one better than control objective actinonin.8. The bacteriostatic effect of actinonin and all the compounds were no better than the positive control Gentamicin. Compared to actinonin, the MICs of only BLC-44 against E.coli and K.pneumoniae were better than it.Conclusion: The Ni2+-PDF of E.coli and iron-enriched PDF of S.aureus were obtained by expression and purification and kept high stability and activity. The screening model was established and the inhibition effects of compounds were determined. The results showed that no compound was better than actinonin. The MICs showed that the bacteriostatic effects of all the compounds were not ideal.