Expression And Purification Of Carbapenemase OXA-72 In Pichia Pastoris And Its Properties Study

Objective:To express and purify OXA-72 in Pichia pastoris.A clinical isolate of Acinetobacter Baumanii(isolate 40),which is the first isolate in our country producing carbapenemase OXA-72,was used as template.This is the basis of the further study on its property and function.Methods:First,we tested the MIC of 12 antibiotics to Acinetobacter Baumanii isolate 40 by E-test.We amplified oxa-72 gene by PCR using the extracted genomic DNA of the isolate 40 as a template and using the target primers P1/P2.Test the PCR product with 1%agarose gel electrophoresis(AGE).Double enzyme digest the PCR product and plasmid pGAPZαA with EcoRⅠand KpnⅠ.Then ligate them with T4 ligase to acquire recombinant plasmid pGAPZ-oxa72.Plasmid pGAPZ-oxa72 was transformed into E coli DH5αfor amplification and preservation.PCR was proceeded using recombinant plasmid pGAPZ-oxa72 as template and vector primer,whose product was tested by 1%AGE.The recombinant plasmid was also tested by double enzyme digestion and sequencing.Linearized recombinant plasmid pGAPZ-oxa72 with restrictive enzyme AvrⅡwas transformed to Pichia pastoris GS115 by electroporation.Vacant plasmid pGAPZαA without oxa-72 was also linearized and transformed into GS115 for control.Then the positive transformants were screened by colony PCR with respectively target primers and vector primers.Thallus of positive clone GS115(pGAPZ-oxa72)和GS115(pGAPZαA)was broken with supersound and its protein was extracted to do western blot analysis.YPD plate containing high concentration Zeocin was used to screen positive clones.Separate the thallus and the supernatant with centrifugalization.Nitrocefin test was done respectively with isolate GS115和GS115(pGAPZαtA)as control.We purified OXA-72 with nickel column.Stepwise elution was done by 50mM and 200mM imidazole.SDS-PAGE and Coomassie brilliant blue dyeing was proceeded to test the molecular weight and purity of the recombinant protein.The last,we submitted the freeze-drying power of the purified OXA-72 to Beijing Proteome Research Center to do isoelectric focusing electrophoresis to determine its isoelectric point.Results:The MIC of Imipenem and Meropenem to isolate 40 was both 32μg/ml. Isolate 40 was also resistant to Cefalotin,Cefoperazone,Amoxicillin and Amoxcillin- clavulanic acid.The 1%AGE showed that DNA sequence amplified by PCR from isolate 40 was about 843bp.The PCR product with vector primers and pGAPZ-oxa72 as template was about 1340bp.Two fragments of 840bp and 3kb was obtained from double enzyme digestion,and sequencing showed that it was the same with oxa-72 gene in Genbank(GenBank accession numbers AY739646).One positive transformant was obtained with both Western blot and Nitrocefin test positive.SDS-PAGE showed the relative molecular weight of the recombinant protein was during 34kDa-43kDa.Western blotting showed the recombinant protein could specifically bind to His-tag antibody.The recombinant protein was obtained by purification with 95%final purity and 40%recovery rate.The isoelectric point of the recombinant protein was 6.37.Conclusion:We successfully expressed and purified the carbapenemase OXA-72, whose isoelectric point was 6.37,in Pichia pastoris,which is the basis of the further study on its property and function.