Expression, Purification and Characterization of Arginase from Helicobacter pylori in Its Apo Form 英文参考文献.docVIP

Expression,PurificationandCharacterizationofArginase fromHelicobacterpyloriinItsApoForm JinyongZhang1,XiaoliZhang1,ChaoWu1,DongshuiLu1,GangGuo1,XuhuMao1,YingZhang2 ,Da- ChengWang2,DefengLi2*,QuanmingZou1* 1Department of Clinical Microbiology and Immunology, Collegeof Medical Laboratory,Third Military Medical University, Chongqing, China, 2National Laboratory of Biomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences,Beijing,China Abstract Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animalsandyeast,onlyafeweubacterialarginaseshavebeencharacterized,suchasthosefromH.pyloriandB.anthracis. 2+ However,theseenzymesusedforarginaseactivityassaywereallexpressedwithLBmedium,aslowconcentrationofMn 2+ was detectable in the medium, protein obtained were partially Mn bonded, which may affect the results of arginase activityassay.Inthepresentstudy,H.pyloriarginase(RocF)wasexpressedinaMn2+andCo freeminimalmedium,the resultingproteinwaspurifiedthroughaffinityandgelfiltrationchromatographyandtheapo-formofRocFwasconfirmed byflamephotometryanalysis.Gelfiltrationindicatesthattheenzymeexistsasmonomerinsolution,whichwasuniqueas comparedwithhomologousenzymes.Arginaseactivityassayrevealedthatapo-RocFhadanacidicpHoptimumof6.4and 2+ 2+ 2+ 2+ exhibited metal preference of Co .Ni .Mn . We also confirmed that heat-activation and reducing regents have significantimpactonarginaseactivityofRocF,andinhibitsS-(2-boronoethyl)-L-Cysteine(BEC)andNv-hydroxy-nor-Arginine (nor-NOHA)inhibittheactivityofRocFinadose-dependentmanner. Citation:ZhangJ,ZhangX,WuC,LuD,GuoG,etal.(2011)Expression,PurificationandCharacterizationofArginasefromHelicobacterpyloriinItsApoForm.PLoS ONE6(10):e26205.doi:10.1371/journal.pone.0026205 Editor:VladimirN.Uversky,UniversityofSouthFloridaCollegeofMedicine,UnitedStatesofAmeri