Studies On The Catalytic Mechanism Of Secreted Phospholipase A2 GⅡE Based On Its Crystal Structure

Objective:1.Express the secretory phospholipase A2（secretedphospholipase A2,sPLA2）GⅡE in Pichia pastoris eukaryotic expression system.Using enzyme and other methods to compare the expression level of BSM culture medium and YPD culture medium to optimize and enhance the expression effect of sPLA2 GⅡE.Compare the expression levels of sPLA2 GⅡE in BSM medium and YPD medium to optimize and enhance the expression level of sPLA2 GⅡE.2.Illustrate the calcium binding mechanism of sPLA2 GⅡE by site directed mutation,enzyme activity determination,calcium affinity experiment and structure simulation experiment.Methods:In this study,the E.coli prokaryotic expression system and the Pichia pastoris eukaryotic expression system were used to recombinantly express sPLA2 GⅡE protein.Purification procedures such as ion exchange,affinity chromatography,and molecular exclusion were used to obtain the target protein with a purity of more than 95%and homogeneous state.Two different media were used to express the proteins of interest to optimize the expression levels of elevated sPLA2 GⅡE.Designed and expressed h GⅡE Mutants.Using the sPLA2 assay kit,the enzymatic activity of different types of sPLA2proteins was measured,and the percentage of enzymatic activity was calculated.Calcium binding dissociation was measured using the Biacore T200 system,and the equilibrium constant（KD）was calculated.Molecular modeling techniques were used to construct sPLA2 GⅡE mutant models using Web Swiss-Model,and reasonable models were selected for analysis.This study was divided into four parts:Protein expression optimization and purification,Enzymatic experiments,Calcium binding assay,and Molecular modeling.1.Protein expression purification:Primers were designed according to the sPLA2 GⅡE protein nucleic acid sequence,and the gene of interest was amplified by PCR reaction;Construction into the yeast cell expression vector p GAPZαA;Transformed E.coli TOP10 competent cells;Recombinant plasmids were extracted,linearized using the Bln I restriction enzyme and electrotransferred into yeast cells;The positive clones were picked and cultured in YPD liquid medium（containing 100μg/ml Zeocin）were expanded to the tertiary species by step culture,then maintained in BSM medium at 200 rpm for 55-60 h at 28℃.During the culture,samples were taken every 6 h to determine the OD value and enzyme activity.The obtained culture was centrifuged at 10000 rpm for 5 min at 4℃,and the pellet was discarded to obtain the supernatant;With The supernatant was filter sterilized by a filter membrane of 0.45μM;Concentrated with a Vivaflow 200 system;Diluted 4-fold with buffer（10 m M MES p H 6.0）.The samples were initially purified using a SP column;Samples were collected according to the UV receiving peak,run on for SDS-PAGE verification,and collected according to the SDS-PAGE gel results.In the purification process,samples were collected at each step,and the protein expression was detected by SDS-PAGE gel running verification and sPLA2 enzyme activity assay kit.Protein purification yields were assessed.2.Enzymatic experiments:The sPLA2 GⅡE mutants was constructed.The changes of protease activities of different mutant types were observed by enzyme activity assay and calcium binding dissociation constant assay.SPLA2 GⅡE wild type and mutant were determined by sPLA2enzyme activity assay kit（Cayman,Ann Arbor,USA）.The substrate of the kit is 1,2-dithioheptyl lecithin analogue（DTPC）.When sPLA2 reacts with the substrate,the sulfur ester bond at sn-2 will be hydrolyzed,and the negatively charged sulfur atom will react with DTNB to develop color.The absorbed light can be detected at the wavelength of 414nm.3.Calcium binding assay:Approximately 4000-5000 reaction units（RU）of purified WT sPLA2 GⅡE and the 3 mutant proteins were immobilized on a CM5 sensor chip.Measurements were performed at a flow rate of 10 m L/min in a Ca Cl₂free HBS running buffer,and temperature 25℃.1 M Ca Cl₂was diluted with running buffer to give a final concentration of 3.125μM,12.5μM,50μM,200μM,800μM and 3200μM。A reference cell without immobilized protein was used as a nonspecific binding control.Data were collected,and equilibrium constants（KD）were calculated with the"affinity"model in Biacore T200software,version 3.1.4.Molecular modeling:sPLA2 GⅡE mutant models were built with Web Swiss-Model.Using the Discovery Studio software package version 3.5,the constructed model was solved in explicit solvent.Further energy minimization was performed to remove geometric restraints of any residue using the steepest descent algorithm.The maximum number of steps was set as 1000,and the final RMS gradient was less than 0.1 kcal/（mol x（?））.No significant differences between the models were found before and after simulation.The reasonable models were selected for analysis.Results:1.SPLA2 GⅡE was successfully expressed by yeast eukaryotic expression system;Through the optimization of culture medium,the protein was expressed in BSM medium,and the protein with high purity was obtained by ion exchange and molecular chromatography,and the obtained sPLA2 GⅡE had good enzyme activity.2.The mutants of sPLA2 GⅡE were successfully constructed,and the enzyme activity of the mutants decreased to various degrees compared with that of the wild-type GⅡE.Among them,16E,N21G-D22F and 16E-N21G-D22F decreased to 59.1%,10.62%and9.13%respectively compared with wild-type sPLA2 GⅡE.3.The results of calcium binding dissociation experiments showed that WT h GⅡE showed calcium binding ability,KD was 0.50 mmol/L,the calcium affinity of three mutants 16E decreased to 2.76-fold,N21G-D22F and 16E-N21G-D22F decreased to10.12-fold and 5.44-fold respectively.4.Compared with WT GⅡE,there was no significant change in the Ca2 binding site of GⅡE mutant 16E.Contrary to the hypothesis that Ca2 can be stable in GⅡE,there is no Ca2 and no Ca2 binding in the models of mutant N21G-D22F and mutant 16E-N21G-D22F.Conclusion:Ca2 plays an important role for the enzyme activity mechanism of secretory phospholipase A2 GⅡE.During the reaction,Ca2 indirectly participated in the catalysis as a backup of Ca1.