大腸桿菌af1514蛋白的分離純化及結(jié)晶工藝的優(yōu)化

大腸桿菌af1514蛋白的分離純化及結(jié)晶工藝的優(yōu)化

分配分配的復(fù)合遺傳技術(shù)的二甲基氧化物和二維表面活性劑的數(shù)據(jù)集是落后的，二級表面活性劑的數(shù)據(jù)集是落后的。正因?yàn)槿绱?，分配分配的貨艙的信息。貨艙的基本信息是分配的。貨艙的基本信息是分配的。另一方面，貨艙的基本信息是不確定的。其他文本中的硫醇鈉函數(shù)（hp）不顯示新的有序排列，而是顯示新的有序排列。Archaeoglobusfulgidusisthefirstsulphate-reducingmicroorganism,whosegenomesequencehasbeendetermined,anditisastrictlyanaerobic,hyperthermophilic,sulfate-reducing,marinearchaeon.Theorganismthrivesinanextremeanddynamicenvironmentlocatedattheinterfacebetweensuperheatedanaerobicventfluidsandfrigidaerobicseawater.A.fulgidushasbeenstudiedwidelysinceitsdiscoverytenyearsago.Itscompletedgenomesequenceoftheorganismprovidesawealthofnewinformationabouthowthisunusualorganismexploitsitsenvironment.Itsgenomeof2178400basepairscontains2436openreadingframes(ORFs).Amongthe2436openreadingframes(ORFs),overhalfoftheA.fulgidusORFs(1290)havenoassignedbiologicalrole.Ofthese,639havenodatabasematch.Theremaining651ORFs,designated‘onservedhypotheticalproteins’,encodesfunctionallyuncharacterizedyetconservedproteins.StructuralstudyofthesehypotheticalproteinswillprobablyaddtoourunderstandingofthegeneticrepertoireoftheArchaea.Solutionofmoreandmorearchaealprotein`sstructurewillprovidenewinformationfortheunderstandingofthefunctionoftheseunkowngenes,aswellasbetterunderstandingofprokaryoticdiversity.Open-readingframeORF-1514inthegenomeofthehyperthermophilicarchaeonArchaeoglobusfulgidusDSM4304encodesa91-residueprotein(GenBankaccessionNo.NP_070343)ofunknownfunctionwithamolecularweightof10.5D.Sequenceanalysis(proteinsequenceMEIMDEIKVNLQKEVSLEEAERYAKNIASKYGDGILLSVHDSKTGYRAPEVYCCGEKPWEVYACNRGANLKISVNQFEFYFRIEVEGQAKY)byWuBlastshowedthatAF1514proteinhasnoalignmentinthePDB(Evalue>1).Sowehavechosenitforstructuralstudiesaimedatcharacterizingitsstructureandidentifyingitspossiblefunction.1會計與方法1.1sofficicicicicicicicicicicidisi最interficicicicicicicicicicicicicicicidisi最參數(shù),socivicicicicicicicicicicicicicicicicicicidisi最參數(shù),socificicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicicidisissicidisi反應(yīng)總結(jié)構(gòu)數(shù),sonicicicicicicicicicicicidisi反應(yīng)總要求solificidicidisi反應(yīng)re堅持colicidisi活itysolificicicicicicicicidisi活結(jié)構(gòu)—ProteinproductionThehypotheticalproteinAF1514fromArcheoglobusfulgiduswasexpressedinEscherichiacolistrainBL21-DE3(Invitrogen)withTEV(tobaccoetchvirusprotease)cleavingN-terminalhexa-His-tagandMBP(maltosebindingprotein).TheglycerolstocksofclonewasinoculatedtoLBmediumcontaining100mg/mlampicillinandgrownat37℃temperaturewhileshakingat200r/minfor2~4h,whenODreached0.6~0.8,theculturewascooledto4℃andthensupplementedwith0.2mmol/Lisopropyl-dthiogalactopyranoside(IPTG)forinduction.Aftersometimeoffermentation(20hat16℃,40hat12℃),thecellswereharvestedbycentrifugationat4000r/minfor30minandsuspendedin40mlPBS(pH7.0)sonicationbuffer.Thecellsweredisruptedbythreecyclesofsonicationeachconsistingofa10spulsefollowedby10sstop.Thecelldebriswasseparatedbycentrifugationat16000×gfor30minandthesolublefractioncontainingtargetproteinwascollectedforfurtherpurification.1.2elulazele.4.3.4和lag-12-coluhn和lag-12-coluhn.3.4和lage-8.3.SupernatantaftercentrifugationwasappliedontoaNi2+-affinitycolumn(Ni-NTAagarose,Qiagen)equilibratedwithPBSbuffer(pH7.0).After30~60minNi-bindingat4℃,thecolumnwaswashedwithwashingbuffer(PBScontaining10mmol/Limidazole),finallyelutedwith10ml500mmol/Limidazole(inPBSbufferpH7.0)atroomtemperature(about20℃).Theelutedproteininimidazolewastreatedtobufferexchange(PBS)byusingconcentratorAmiconUltra-45000MWCO(Millipore)andthendigestedwithTEVat30℃for3horat4℃overnight.Afterdigestion,thesupernatantpassedthroughNi2+-affinitycolumn(Ni-NTAagarose,Qiagen)onceagaintoremoveN-terminalHis-tagandMBP.Theflowthroughcontainingtargetproteinwasconcentratedto1mlbyultra-filtration(AmiconUltra-45kDMillipore)andfurtherpurifiedbygelfiltration.20~30μlofsamplewastakenaftereachpurificationstepanddetectedby12%~15%SDS-AGE(load3~5μlsample).1.3ershpo對于trsis的預(yù)測ConcentratedproteinsamplewasappliedontoSuperdex7510/30gel-filtrationcolumn(AmershamBiosciences)equilibratedwithcrystallizationbuffercontaining200mmol/LTris,100mmol/LNaCl,and1mmol/LDTTforthefurtherpurification.Theidentityandpurityofthepreparedsamplewasjudgedby15%SDS-AGEstainedwithCoomassieBrilliantBlue.1.4e主要產(chǎn)物的so階段Thepurifiedproteinwaschemicallymodifiedusingareductivemethylationprotocolasdescribedpreviously.20μl1mol/Ldimethylamine-boranecomplex(DMAB)and40μ1mol/Lformaldehydewereaddedto1ml10μg/mlproteinsolution.Thereactionmixturewasincubatedinthedarkwhileshakingat220r/min.Thechemicaladditionswererepeatedtwiceat2hintervals.Finally,10μl1mol/Ldimethylamineboranecomplex(DMAB)wasaddedandthereactionmixturewasincubatedovernightat-4℃.Excesschemicalswereremovedbysize-exclusionchromatographyusing200mmol/LTris,100mmol/LNaCl,and1mmol/LDTTbuffersolution.Themethylatedproteinwasconcentratedto15~20mg/mlandusedforcrystallizationsimilartothenonmethylated(native)protein.1.5機(jī)械scening成u2004相關(guān)文獻(xiàn)Proteinobtainedbygelfiltrationwascrystallizedbythehanging-dropvapour-diffusionmethodat16℃.1μldropletofproteinsolutionmixedwiththesameamountofreservoirsolutionwasequilibratedagainst300mlreservoirsolution.TheinitialcrystallizationconditionswereexaminedusingcommerciallyavailablescreeningkitsfromHamptonResearch(CrystalScreen1,CrystalScreen2,IndexandPEG/IonScreen)andEmeraldBiosystems(WizardIandII).Becauseoftheimportanceofgoodcrystalshapeandbetterdiffractionqualityinproteincrystallographicstudy,thecrystallizationconditionsneedfurtheroptimizingafterpreliminarycrystallizationstudy.Goodcrystalsappearingunderdifferentconditionswereharvestedincryoloopsandsoakedfor1mininparaphinesolution.Thecrystalmountingincryoloopwasash-cooledinnitrogen-gasstreamat_196℃.DiffractionpatternsofthecrystalsweredetectedusingaX-rayRAXIS4detectorwithCrresourceattheRIGAKUdemoX-raylaboratory,InstitutionofBiophysics,CAS.2影響的分析2.1u3000et.4.3.4重組病毒meq指數(shù)etinTargetproteinwithmolecularweightof54(containedN-terminalhexa-his-tag&MBP)canbedetectedonSDS(Fig.1).Expressionoftheproteinisveryhigh.Aftercentrifugation,mostofthetargetproteinexistsinsupernatant,anditisclearthattheproteinAF1514issoluble.Resultsofthestudyalsoshowedthatinducedproteinproductionismuchhigheratlowertemperature,andforlongerfermentationtime(12℃for40h).Theproteinpurificationstepwasconductedatroomtemperature(20~25℃)andAF1514seemstobenotverysensitivetotemperature.2.2tevtraftingslaft計劃ThealmostpuretargetproteincanbeobtainedafterpurificationwithNi-bindingaffinitycolumn.Althoughsomeofthetargetproteincanbewashedoutwith10mmol/Limidazolewashingbuffer,stilldesirableamountofthetargeproteinisbindwelltothecolumnandcanbeelutedwith500mmol/Limidazole(Fig.2).Forhighthroughputcloningandexpressionoftargetproteins,theclonedtargetproteinincludedapolyhistidineaffinitypurificationsequence(His-tag),themaltosebindingprotein(MBP),whichimprovesthesolubilityofexpressedproteinsandarecognitionsequenceforthehighlyspecifictobaccoetchvirusprotease(TEV-site).ThereforeTEVtreatmentisnecessaryforthefurtherpurification.ThecleavageofMBPandhexa-HistagfromtargetproteinwithTEVproteaseisbetterat4℃overnightthanat30℃for3h(Fig.2).AfterTEVtreatment,proteinAF1514withMWof10.5kDcanbesuccessfullyseparatedfromHis-tagandMBPbysecondNi-affinitychromatography(Fig.3).2.3表面活性劑競爭參數(shù)Gelfiltrationisthesimplestandmildestamongallthechromatograhymethodsusedforproteinpurification.Targetproteinwaspurifiedbygelfiltrationusingbuffercontaining200mmol/LTris,100mmol/LNaCland1mmol/LDTTatpH7.0.AsinglebandcorrespondingtothehypotheticalproteinAF1514(91residuesandmolecularweight10.5kD)wasobservedaftersizeexclusion(Fig.4).Becausesuitableproteinconcentrationisimportantincrystalgrowth,theconcentrationofthefinalpureprotein(45.8mg/ml)wasdetectedbyspectrophotometerat280nmanddilutedto15~20mg/mlwithsamebufferbeforesettingupthecrystals.2.4kimace.3.4與主要前藥分子的關(guān)系Crystalsgrowedundersomeoftheconditionsoftotal432differentconditionsusedforcrystalscreen(Table1).Fornativeprotein,crystalsappearwithin2~3dofincubationunderseveralconditionssuchasCrystalScreen1-condition24,PEG/Ion-conditon48andMembFacconditionNo.1.Butthecrystalgrowthofthenative(nonmethylated)proteinandmethylatedproteinaredifferentunderthesescreenedconditions.CrystalshapeisimprovedinmethylatedproteinunderCrystalScreen1-condition24andPEG/Ion-conditon48inwhichnativecrystalgrowthisn`tgoodinshapeornotgrowsatall.Thesameshapeofcrystals(tetraganol)growsunderMembFaccondition1forbothnonmethylatednativeproteinandmethylatedprotein(Fig.5).OneofthemostpromisingcrystallizationconditionMembFaccondition1wasfurtheroptimizedinordertoobtaingooddiffiractionpatternsoftheproteincrystals.TheconditionwasfurtherrefinedbychangingthepH(4.0,4.5and5.0)ofthebufferandtheMPDconcentration(10%~20%).FinallythediffractionpatternsofthecrystalsweretestedinacoupleofdaysafteritsgrowthusingX-raydetector(RAXIS4,Rigaku)andthecrystalswhichhavebetterdiffractions(resolution2.09Ao)appearonlyunderoptimizedconditionsofMembFacconditionNo.1(0.1mol/LsodiumacetatepH5.0,0.1mol/Lsodiumchloride,8%~14%(W/V)2-methyl-2,4-pentanediol(MPD)).(Dataarenotshown).3u2004范圍Allorganismscontainthousandsofdifferentproteins,whichplayessentialrolesinmaintainingtheirlife.Aprotein`sstructuredeterminesthespecificrolethatitplaysintheorganism.However,researcherslackdetailedknowledgeaboutthestructuresofmanyproteins.Crystallographyallowsscientists,throughthestudyofproteincrystals,todeterminethethree-dimensionalmolecularstructuresofproteins.Forthis,scientistsmustfirstcrystallizetheproteinandanalyzetheresultingcrystalsbyX-raydiffraction.Thestructuresofmanyimportantproteinsremainamysterysimplybecauseitisdifficulttoobtaincrystalsofhighenoughqualityorlargeenoughsizeandtheproteinmoleculesarranginginanorderlyandrepeatingpattern.Withoutperfectcrystalsofprotein(oranyotherbiologicalsamples)itisimpossibletocarryoutanycrystallographicstructuralstudies.InordertounderstandthefunctionsoftheproteinAF1514basedonitsstructures,wecarriedonpreliminarycrystallizationstudytotheproteinAF1514fromArcheoglobusfulgidusDSM4304andfinallygotgoodcrystalssuitableforstructuralstudybyX-ray.TheopenreadingframeofAF1514consistsof273bpcodingfor91amino-acidresidues.Theisoelectricpointofthetargetproteiniscalculatedtobe5.17.ProteinAF1514canbepurifiedintwostepsto95%puritybyNi2+-affinitycolumnandSuperdex-75gelfiltration.Theresultsofpurificationprocedureshowedthattheproteinseemsnotverysensitivetothetemperature.Amother-liquorsolutioncontaining