TwovariantsofAffinityTagsforPurification:TandemAffinityTag&Self-cleavingpurificationtags匯報人:宋健Cellreview-2013-FongB.A.-Thepotentialroleofself-cleavingpurificationtagsincommercial-scalesprocess2015.06.03TwovariantsofAffinityTagsforPu11Background2

TandemAffinityTag3Self-cleavingpurificationtags4ConclusionsandInspiration1Background21Background1.11Background1.13Traditionally,apurificationtaghasimplieduseofanaffinityseparationmethod.Withthesemethods,theaffinitytagisexpressedasafusionpartnerwiththedesiredtarget.Thetagbindsstronglyandselectivelytoanimmobilizedligandonasolidsupport,andcellandprocesscontaminantsarewashedaway.Affinitychromatographytypicallyyieldspurities>90%inasinglecolumnstep.1.2AffinityTagsTraditionally,apurification4(DYKDDDDK)EDTAWSHPQFEK(鏈親和素)6HIMAC(immobilizedmetal-ionaffinitychromatography)ImidazoleSLAELLNAGLGGSandTKDPSRVGmildconditiongoodforcomplexes1.2.1CommonAffinityTags(DYKDDDDK)EDTAWSHPQFEK5基于親及層析兩種蛋白質純化策略6基于親及層析兩種蛋白質純化策略7Fig.1.Differentstrategiestoincorporateandremoveaffinitytags.1.2.2ConstructionsFig.1.Differentstrategiest8Con11.2.2ConstructionsCon1：alinkerregionincludingaspecificsequenceforendoproteasecleavageincreaseaccessibilityoftheaffinitytagandisoftenrequiredforeffectiveendoproteasecleavageCon11.2.2ConstructionsCon1：a91.2.2ConstructionsCon2Con2：aidsolubilityandfoldinglikemaltose-bindingprotein(MBP),glutathioneS-transferase(GST)orsmallubiquitinmodifyingprotein(SUMO),thioredoxin(Trx),amongothers.SometagssuchasMBPorGSTareusedforbothaffinitypurificationandsolubility.GSTbindingglutathione-Sepharoseresinwiththeslowbindingkineticsistimeconsuming.MBPcanbepurifiedonamylosebutmayresultinproteindegradation.Histagbothnativeanddenaturingconditionscanbeusedduringpurification1.2.2ConstructionsCon2Con2：a101.2.2ConstructionsCon3Con3：thesimplestgeneticdesign-afusionproteindesignedforexopeptidaseremovalofthetag(onlyforN-terminaltags,usingTAGZyme)1.2.2ConstructionsCon3Con3：t111.2.2ConstructionsCon4Con4：afusionproteinwhereasolubilityandfoldingpartnerisfusedN-terminaltothetargetprotein(e.g.,SUMO,sortaseA).AnaffinitytagattheN-terminusisrequiredforpurification.SUMOhasemergedasanalternativefortheproduction,solubilityandcorrectfoldingofotherwiseintractableproteins.TheSUMOtagcanberemovedusingaspecificprotease(e.g.,theyeastSUMOprotease-1Ulp1)thatrecognizestheconformationoftheubiquitinpartnerratherthanaspecificsequence.TheuseofSUMOismostlyconstrainedtoE.coli,sincehighlyconservedSUMOproteasesarepresentineukaryotes.1.2.2ConstructionsCon4Con4：a12(i)improveproteinyield(ii)preventproteolysis(iii)facilitateproteinrefolding(iv)protecttheantigenicityofthefusionprotein(v)increasesolubilityAffinitytagshavealsobeenusedtoincreasethesensitivityofbindingassaysfortaggedScFv(單鏈抗體single-chainantibodyfragment,scFv)1.2.2Benefits(i)improveproteinyield1.2.13(i)achangeinproteinconformation(ii)lowerproteinyields(iii)inhibitionofenzymeactivity(iv)alterationinbiologicalactivity(v)undesiredflexibilityinstructuralstudies(vi)toxicity1.2.3Negativeeffects(i)achangeinproteinconfor14Inmanyscenarios,thepresenceofapurificationtagmightaltertheactivityofthetargetprotein,orcauseatherapeuticproteintobecomeimmunogenic.Thus,tagremovalisacrucialrequirementformanyofthesemethods.Thefollowingsectionreviewsvarioustagremovalstrategiesandhowimprovementsinthesemethodsmighthaveanimpactonfutureuse.1.3TagremovalmethodsInmanyscenarios,thepresenc15Proteasesareenzymesthatcleaveproteinsatspecificsequencesandarethereforepotentialcandidatestocatalyzetagremoval.Exopeptidases(aminopeptidasesandcarboxypeptidases)catalyzetheremovalofaminoacidsfromtheendofaproteinandcanbeusedfortheremovalofverysmalltagssuchasHis6.SeveralaffinitytaggedprocessendoproteasesarethereforeusedfortagremovalsuchasThrombin,virus-derivedTEVprotease,3Cprotease,GranzymeBandCaspase-6,usedfortheremovalofHis6,GST,FLAG.1.3.1ProteasesProteasesareenzymesthatcle16Ageneraldrawbackofendoprotease-mediatedtagcleavageistheneedforhighratiosofenzymetoproteinandlongincubationtimerequiredforcompletetagremoval.Animportantlimitationisthathighlyspecificendopeptidasesaretypicallyexpensiveandunavailableinquantities.Inaddition,manyoftheseproteasesarehighlybioactive(e.g.thrombin凝血酶andFactorXa)andmustbeseparatedcompletelyfromthetargetprotein,oftenbyanadditionalpurificationstep.withoutrequiringaspecificsequenceattheC-terminusofthecleavagesiteacysteinepeptidasethatsequentiallyremovesdipeptidesfromtheN-terminusuntilastoppositionAgeneraldrawbackofendoprot17Theprocessisdesignedfortheinitialpurificationofthehis-tagproteinusingIMAC;atagremovalstepwhereaprotease(orpeptidase)—thatalsocontainsahis-tag—isaddedtothehis-tagproteinandafinalsubtractiveIMACpurification.Inthislaststep,processimpuritiessuchasunprocessedhis-tagprotein,theprotease(orpeptidase)andanyunspecificbinderareretainedinthecolumnwhilethedetagged‘pure’proteiniseluted.Fig.2.Overviewofaprocessforthepurificationofhis-tagproteinsandsubsequentlytagremoval.1ststep2ndstep3rdstepTheprocessisdesignedforth18Inteinsareself-splicingproteinelementsthathavetheuniqueabilitytoexcisethemselvesfromwithinatranslatedhostprotein.Self-splicinginteinscanbemutatedatN-orC-terminalsplicejunctionstoyieldself-cleavinginteins,whichcanthenbeusedtomediateself-cleavingofvariouspurificationtags.Theseinteinscanbesplitintotwocategories:thosewithpH-inducedC-terminalcleavingactivity,andthosewiththiol-inducedN-andC-terminalcleavingactivity.1.3.2Inteins（內含肽）Inteinsareself-splicingprot191.3.3Comparisonbetweenproteasesandinteins1.3.3Comparisonbetweenprote202Self-cleavingpurificationtagsFig.3.Figure1.Varioustagremovalmethodsandkeyresidues:(a)pH-inducedinteins;(b)thiol-inducedintein;(c)FrpCprotein;(d)sortaseAtranspeptidase(SrtAc);and(e)proteases.2Self-cleavingpurificationt21內含肽剪接原理4步親和反應：1.N-S酰基重排；2.轉脂反應；3.Asn環化；4.N-S酰基重排。內含肽剪接原理4步親和反應：22FIGURE1.Self-cleavingaffinitytagsbasedoninteins.A,TheIMPACT-CNsystem(left)includesanaffinitytagwithintheintein,andC-terminalcleavingisinducedviathiol-inducedcleavageofashortN-terminalexteinpeptide(step1atbottom).CleavageofN-exteinpeptidethenleadstorapidC-terminalcleavage(step2atbottom),andtheN-terminalpeptideissubsequentlyseparatedfromthecleavedtargetproteinbydialysis.TheΔI-CMintein(right)providesC-terminalcleavingintheabsenceofN-terminalcleavage,wherethecleavagereactioniscontrolledbyshiftsinpHand/ortemperature.Inthisintein,thecleavagereactionisadditionallyacceleratedthroughmutationofaconservedasparticacidtoglycine,closetotheCterminusoftheintein.cFIGURE1.Self-cleavingaffini23B,theSspDnaB（解旋酶）inteinhasbeenengineeredwithan11-residuedeletionatitsNterminustoeliminateprematurecleavingduringexpression.C-terminalcleavageoftheinteincanbeinducedbytheadditionofthe11-residueinteinsegment(leftpanel),orconversely,N-terminalcleavingfromthe11-residueinteinsegmentcanbeinducedbytheadditionoftheremaininginteininthepresenceofthiol(rightpanel).B,theSspDnaB（解旋酶）inteinha24C,theNpuDnaEnaturallysplitinteinhasbeenengineeredwithaninternalaffinitytagtoprovideextraordinarilyrapidcleavinguponreassembly.ThisinteineffectivelycombinestheN-exteinremovaloftheIMPACT-CN

systemwiththeasparticacidtoglycinemutationoftheΔI-CMintein,leadingtoveryrapidcleavingthatcanbecontrolledbyinteinreassemblyinthepresenceofzinc.進步意義：降低溫度：4℃可行；節約時間：室溫下30minC,theNpuDnaEnaturallyspli25FrpCundergoesaself-cleavingreactionbetweenresiduesAsp414andPro415uponcalciumionaddition.ThisdomainhasbeenpairedwithCBDorHistagstomakethemeffectivelyself-cleaving.FrpCcleavageistightlycontrolled,withnearlynoprematurecleavageduringproteinexpression.Additionalneeds:10mMDTT;EDTA.2.2.1FrpC腦膜炎雙球菌提取蛋白FrpCundergoesaself-cleaving26SortaseAtranspeptidase(SrtAc轉肽酶)thatwasdiscoveredinthecellenvelopeofStaphylococcusaureus（金黃色葡萄球菌）.FusionofthecatalyticcoreofSrtActoitsaminoacidrecognitionsequenceLPETGgeneratesaself-cleavingmodule.Inthiscase,theThr–GlybondwithintheLPETGsequenceiscleavedinthepresenceof5mMCa2+and5mMtriglycine,whicheffectivelycleavesthetargetproteinfromtheC-terminusoftherecognitionsequence.2.2.2SrtAcSortaseAtranspeptidase(SrtA27ChitinBindingDomain(CBD)2.3內含肽在蛋白純化中的應用ChitinBindingDomain(CBD)2.28Elastin-likepolypeptide(ELP)彈性蛋白樣多肽特性：溫度T＞30-40℃不溶

降低溫度，低pH條件下可重懸與ELP聯用Elastin-likepolypeptide(ELP)29與PHB聯用PHB(polyhydroxybutyrate)granules（聚羥基丁酸酯顆粒）PHA：聚羥基脂肪酸酯、植物血凝素，是一種多聚糖，在大多數原核生物中表達為顆粒狀包涵體，PHB為其主要的聚集形式。Phasins：PHB調節蛋白，可特異性吸附于PHB顆粒表面，高親和力。與PHB聯用PHB(polyhydroxybutyrate30內含肽其他應用內含肽其他應用313TandemAffinityTag鈣離子螯合劑鈣調素結合多肽煙草蝕紋病毒蛋白酶3TandemAffinityTag鈣離子螯合劑鈣調32

4ConclusionsandInspiration1.Self-cleavingtagshavethepotentialtohaveasignificantimpactonproteinpurificationinthecommodityenzymeandhumantherapeutic