Overexpression And Properties Analysis Of Inorganic Pyrophosphatase Overexpressed In E.COLI

Inorganic pyrophosphatase(PPase, EC3.6.1.1) are essential enzymes that are important incontrolling the cellular concentration of inorganic pyrophosphate. It can catalyze thehydrolysis of one molecule of pyrophosphate into two phosphate ions.. In the living cell,pyrophosphate is a by-product generated by the NTP/dNTP involved biosynthesis，such asnucleic acid, pyrophosphate will be degarded rapidly by pyrophosphatase, which reduce thenegative effect caused by pyrophosphate accumulation. Using thermophilic DNA polymerasethe PCR reaction simulate the DNA biosynthesis process without the pyrophosphatedegradation. whether the accumulation of pyrophosphate will affect the amplificationefficiency of DNA polymerase? And whether inclusion phosphatase can improve theamplification efficiency of PCR? These questions need to be clarified and analyzed. In thiscommunication, we constructed the prokaryotic expression vectors of inorganicpyrophosphatase, and obtained the optimum expression condition and analyzed thecharacteristics of the catalytic activity of rPPase.The PPase coding sequence was amplified from the genomic DNA of T. thermophilusHB8by PCR and sequenced. The PPase sequence showed100%identities compared with T.thermophiluss PPase gene reported by using BLASTN. We successfully built its prokaryoticexpression vector pET28a-PPase. The molecular weight of rPPase expressed in E.coli BL21star(DE3) was about20.3kDa. The rPPase was purified through the Talon resin and itsoptimum expression condition were37℃,0.8mmol/L IPTG with8h induction. And totallyobtained50mg of purified enzyme from1L cultures.The enzymatic compare energy was about140U/mL under the standard assay conditions,The optimum pH and temperature of rPPase was8.0and65℃seperately. And its parameterKmwas1.69×10-2mol/L，Vmaxwas13.16mol-1·L·s-1.By using the method of semi-quantitaitve PCR and quantitative PCR, we found that theactivity of the seven kinds of DNA polymerases were inhibited by PPi in PCR system. Afteradding rPPase into the system, the activity of DNA polymerase could recover its activity.Also it could enhance the PCR products and the activity of iProof, Pfu, Pfu turbo, S-KT andS-KOD DNA polymerase through using qPCR while the low concentration of the template.As a result, rPPase could be a new PCR enhancer for these five kinds of DNApolymerase.The experiment showed us the catalytic properites of rPPase through constructing theprokaryotic expression vectors, in order to realise the mechanism of PPase in vivo, anddemonstrate the feasibility whether rPPase could be a new PCR enhancer. This study providetheoretical basis for the application of commercial, and also lay the foundation for the studyof machnism of phosphatase in the body.