System Construction And Optimization Of PBPm-MDCC ATPase Activity Assay And Its Application In Pif1 Helicase

ATPase activity is an important index to measure the activity of many proteins,especially cell metabolism-related enzymes.Effective ATPase activity assays are essential in molecular biology research.The ATPase activity of the enzyme can be calculated by directly detecting the change of ATP concentration in the solution or by measuring the change of Pi concentration produced after the hydrolysis of ATP.In the latter method,a more mature method is PBPm-MDCC ATPase activity assay system.The basic idea is to use fluorescence-labeled periplasmic phosphate binding protein(PBP)as a probe to detect the characteristics of fluorescence signal change caused by the conformation change of PBP and Pi,and to detect the ATP hydrolysis released Pi,thereby achieving the measurement of ATPase activity.This method,which was established in 1994,has high sensitivity and can be used for real-time detection.After more than 20 years of improvement and optimization,it has gradually become one important methods for detecting ATPase activity in molecular biology.The successful construction of PBPm-MDCC ATPase activity assay system mainly depends on three aspects: The cost-effective acquisition of PBPm protein and PNPase protein(purine nucleoside phosphorylase).The effective labeling of PBPm by MDCC and purification of labeled PBPm.The removal of free inorganic Pi from ATPase activity assay system.In previous studies,the methods for the expression and purification of PBPm protein and PNPase protein were cumbersome,time-consuming and laborious.The labeled PBPm protein with fluorescent dye MDCC could not be separated effectively and the final labeling and purification efficiency was low.The free inorganic Pi in the system of ATPase activity measurement was not removed effectively,so that the background value and error were not satisfactory.In order to optimize the assay method of PBPm-MDCC ATPase activity more simply and efficiently,we aimed to develop efficient and practical methods for the expression and purification of PBPm and PNPase proteins,to explore the high efficiency labeling of PBPm by MDCC and the the purification of labeled products,to construct ATPase activity assay system using PBPm-MDCC and Pi-mop,and to verify the feasibility by ScPif1 helicase ATPase activity detection experiments.This study mainly achieved the following results:1)PBPm(A197C)single mutation and PBPm(A17C/A197C)double mutant gene(PhoS)were successfully cloned from E.coli genomic DNA and ligated into pET15b-SUMO expression vector.The induced expression condition and the purification process of PBPm protein were optimized.Finally,more than 135 mg of PBPm protein with a purity of more than 98% could be obtained from one liter of culture.Compared with the literature reports,higher yield,purity,labeling efficiency,material and time cost efficiency were obtained.2)The PNPase gene(deoD)was successfully cloned from E.coli genomic DNA and the E.coli expression vector pET15b-PNPase was constructed for the first time.After induced by IPTG,it was purified in one step using His Trap HP column.More than 170 mg of PNPase protein with a purity above 95% and high activity could be obtained from one liter of culture.The expression and purification method is simple and effective.3)The system conditions of PBPm(A197C)labeling by fluorescent dye MDCC were found,and the active PBPm(A197C)-MDCC was purified by Superdex-200 chromatography column and HiTrap Q column.4)The Pi-mop system was successfully constructed using PNPase protein and 7-methyl guanosine.The Pi-mop could remove the free inorganic Pi from the PBPm-MDCC detection system effectively,and improve the sensitivity of the detection system.The response of PBPm-MDCC detection system to Pi was tested by microplate reader and fluorescence spectrophotometer,and the standard curve of the fluorescence intensity of PBPm-MDCC detection system with Pi was obtained.5)Using the expression vector pET-28 a,we successfully expressed the helicase ScPif1 in Rosetta strain,and purified the ScPif1 protein with purity of more than 95%.Dynamic Light Scattering analysis shows that it exists mainly in the form of monomer in solution and has good uniformity.6)The PBPm-MDCC ATPase activity detection system constructed in this study was successfully applied to the ATPase activity assay experiment of ScPif1 helicase,and the DNA unwinding activity of the ScPif1 with four NTPs was compared.The experiment proved that the system has high practicality and can be used as a simpler and more effective method for the detection of ATPase activity,which can be helpful for the NTPs related research in molecular biology.