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Expression And Purification Of Tyrosine Protease Ltp1 From Porphyromonas Gingivalis

Posted on:2021-03-03Degree:MasterType:Thesis
Country:ChinaCandidate:Q C GuFull Text:PDF
GTID:2404330611458425Subject:Oral and clinical medicine
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Porphyromonas gingivalis,a well-known pathogenic bacteria,is the main cause of periodontal disease,especially chronic periodontitis.Its pathogenic factors are mainly composed of the outer membrane components,such as cilia,lipopolysaccharide and adventitia membrane vesicles,as well as a variety of extracellular proteases,such as endotoxin,phosphatase et al.Tyrosine protease is one of the major virulence factors,but its molecular function remains unclear.Objective: We will construct the target gene Ltp1 from Porphyromonas gingivalis in the prokaryotic expression system,express and purify the protein Ltp1.The purified protein will be used for crystallization.Our aim is to solve the crystal structure of Ltp1 to understand its molecular mechanism and pathogenesis.Materials and methods: The target gene of Ltp1 was synthesized directly,and then was constructed on the p ET29 vector with histidine tag by homologous recombination method.Protein expression in E.coli was induced by isopropyl-?-D-thiogalactoside(IPTG).Afterwards,the protein was purified by nickel affinity chromatography and size-exclusion chromatography.The enzymatic assays will be performed by using the generic substrate of p-Nitrobenzene phosphate.Results:1.In this study,the target gene Ltp1 from Porphyromonas gingivalis was successfully cloned into the p ET29 vector.The protein Ltp1 of 18.75 k Da was expressed in E.coli with the addition of IPTG.2.The expressed protein Ltp1 was confirmed by immunoblotting using the anti-His tag antibody.3.The protein Ltp1 is indeed an active tyrosine phosphatase,which could effectively hydrolyze the generic substrate p-NPP.The optimal reaction temperature andp H were 37-50?,and 5.0-7.0,respectively.Conclusion: The p ET29 vector harboring the gene Ltp1 could express the stable protein Ltp1 in E.coli,as confirmed by western blot.Moreover,Ltp1 could catalyze the hydrolysis of p-NPP,confirming the function as a tyrosine phosphatase.All these results provide the basis for the further structural and functional studies of Ltp1.
Keywords/Search Tags:Porphyromonas gingivalis, protein expression and purification, enzymatic activity, tyrosine phosphatase, periodontal disease
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