Expression And Purification Of PBP1B From Escherichia Coli And PPBP3from Pseudomonas Aeruginosa

Owing to the abuse of antibiotics, bacterial resistance is more and more serious.The multiple drug resistant strains, and even to superbugs resisting almost all antibiotics appeared, making it a serious threat to human health. Escherichia coli and Pseudomonas aeruginosa is two kinds of opportunistic pathogens in clinical, structure change and overproduction of PBP1b and PBP3play important role in the drug resistance. The bifunctional transglycosylase penicillin-binding protein lb (PBPlb) from Escherichia coli and penicillin-binding protein3from Pseudomonas aeruginosa, essential for cell wall synthesis, play vital role in the two bacterias survival. Therefore, in this paper, using genetic engineering methods to synthetize PBP1b and PBP3respectively, to screen new antibacterial drug.To extracted genomic DNA of E. coli CMCC44102and P. aeruginosa CMCC10104with cell genomic DNA Extraction Kit. Designing specific primers of MrcB and ftsl according to E. coli ATCC8739and P. aeruginosa PAO1registered in GenBank, and amplified gene by PCR. After purified by the DNA Gel Purification Kit, the MrcB and ftsl gene fragments were extracted and ligated with pMD19-T simple vector. The recombinant vector were transformed into competent E. coli DH5a. The correct pMD19-T-PBP1b and pMD19-T-PBP3plasmid ware digested with Nde I and Xho I. The PBP1b and PBP3fragment were inserted into appropriately digested pET44b(+) plasmid DNA and transformed into BL21(DE3) strain. It was ready for expressing the recombinant protein with IPTG induction. The results shows that the recombinant genes express better in the condition of100μg/mL Amp,37℃,3h,2mM IPTG.In this expressing condition, PBPlb exists in the form of inclusion body while PBP3is soluble protein.Immobilized metal affinity chromatography (IMAC) with Ni2+-NTA agarose was used for the purification of recombinant PBP1b and PBP3. the balance buffer is composed of20mM PBS,500mM NaCI,20mM imidazole,pH8.0and elution buffer is composed of20mM PBS,500mM NaCI,200mM imidazole,pH8.0.The purified inclusion body PBP1b was renatured by slowly dialysis. The purified proteins were desalted by dialysis and concentrated with ultrafiltration centrifuge tube.Western immunoblotting was performed for further verification of the recombinant PBP1b and PBP3. Using a rabbit anti-His multicloning antibody as the primary antibody and a goat anti-rabbit IgG+horseradish peroxidase(HRP)conjugated antibody as the secondary antibody. The coloration reagent is DAB. It indicated both of them had specific reaction bands.Combined fluorescein isothiocyanate (FITC) with ampicillin (Amp), the products were named FITC-Amp. The purified recombinant protein were able to bind FITC-Amp.Separation and purification by NATIVE-PAGE, the corresponding strip can be seen under the UV lamp of particular wavelength. It shows that recombinant proteins were functional.