Prokaryotic Expression And Enzymatic Assay Of Secretory Phospholipase A₂ Group Ⅲ Of Cyprinus Carpio

secretory phospholipase A2 Group Ⅲ(PLA2g3)is a group of enzymes that liberates the sn-2 fatty of acyl chains from phospholipids to yield nonesterified fatty acids and lysophospholipids.It is involved in a variety of inflammatory reactions,neurosecretion,cell proliferation and atherosclerosis.Vertebrate PLA2g3 is a class of enzymes with unique N-terminal and C-terminal besides catalytic region.At present,there are few reports on the enzymatic activity of PLA2g3 N-terminal and C-terminal.As a worldwide farmed fish,Cyprinus carpio has important research value.In this paper,Cyprinus Carpio was selected as the research object to construct a prokaryotic expression vector which includes PLA2g3,catalytic region(PBVR),N-terminal and C-terminal;an active soluble protein was obtained and enzyme activities of the prokaryotic expressed protein was determined.A large number of protein can be obtained by prokaryotic expression,so that functional analysis of the protein can be performed.However,due to lack of enzymes or cofactors required for accurate folding of eukaryotic proteins in prokaryotic cells,how to obtain soluble recombinant proteins is a bottleneck to be solved in prokaryotic expression system.A tagged protein that promotes soluble expression of target proteins is screened by fusion expression with a tagged protein.This experiment compared the effects of different fusion tags on enhancing soluble prokaryotic protein expression of Cyprinus carpio Group ⅢPhospholipase A2(PLA2g3a1)catalytic active region(PBVR),obtain the soluble prokaryotic recombinant protein of PBVR,and determine the PLA2 activity of PBVR recombinant proteins.The PLA2_bee_venom_like region(PBVR)of Cyprinus carpio PLA2g3a1 was confirmed using BLAST.The PBVR prokaryotic expression vectors were constructed with SUMO、TrxA、NusA、MBP fusion tags,and then plasmids were transformed into E.coil Transetta(DE3)strain to induce the confusion proteins expression.The soluble prokaryotic recombinant proteins were purified by Ni-NTA column and the concentrations of purified proteins were determined by comparison with BSA standard protein.The phospholipase enzymatic characteristics of PBVR recombinant protein were measured using coupling lipoxygenase method.The NusA tag has the highest enhancing soluble effect,followed by MBP,TrxA,and the soluble protein accounted for 95%,87%,and 47%of total protein,respectively.However,MBP fusion protein obtained in unit bacteria was the most.The phospholipase enzymatic activity of MBP-PBVR of was higher than TrxA-PBVR and NusA-PBVR.The optimal pH of MBP-PBVR for catalysis was 7.5.Its phospholipase enzymatic activity has no significant differences among the suitable growth temperatures of Cyprinus carpio.Micro-Ca2+can dramatically improve its activity(Kd=.0.0153(mmol/L)-1).Phospholipid hydrolysis followed classical Michaelis-Menten kinetics(Vm=2.2 μmol/L/min,km=31.9(μmol/L)-1).Results indicate that MBP has a good solubilizing effect,and the purified MBP-PBVR prokaryotic expressed protein has MBP-PBVR phospholipase activity.In order to analyze the effect of N-terminal and C-terminal of PLA2g3 on the enzymatic activity of this protein,prokaryotic expression vectors of pMAL-c2X-N-PBVR and pMAL-c2X-PBVR-C were constructed in this experiment,and soluble prokaryotic recombinant protein was obtained;the phospholipase activity of the protein was determined.Results showed that Amount of MBP-N-PBVR soluble protein and MBP-PBVR-C soluble protein obtained from 1 g total bacteria were 0.375 mg and 0.125 mg respectively.The phospholipase enzymatic activity of MBP-N-PBVR(4.86±0.06μmol/(min·μmol))was lower than that of PBVR(13.68±0.12μmol/(min·μmol)),but MBP-PBVR-C(16.46±0.4μmol/(min·μmol))phospholipase activity was insignificantly higher than PBVR(13.68±0.12μmol/(min·μmol));this result indicates that the presence of the N-terminus of PLA2g3 reduces the phospholipase activity of the protein,and the presence of the C-terminus of PLA2g3 does not affect phospholipase activity.The optimum pH of the enzymatic reaction of MBP-N-PBVR and MBP-PBVR-C was the same as that of MBP-PBVR,both of which were 7.5.10 mmol/L Ca2+can significantly increase phospholipase activities of MBP-N-PBVR and MBP-PBVR-C.This is different from Ca2+promoting the phospholipase activity of MBP-PBVR,showing the presence of N-terminal and the C-terminus delays Ca2+-activated phospholipase activity.Results indicate that the presence of the N-terminal of PLA2g3 reduces the phospholipase activity and delays activation of Ca2+;the presence of the C-terminal does not affect the phospholipase activity,but delays that Ca2+activated phospholipase activity.