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蛋白质保存Thechoiceofstoragemethoddependsonseveralcriteria,includingtheintrinsicstabilityoftheprotein,thedurationofstoragerequired,andhowstringenttheneedforpreventingdamageis.Thereistremendousvariabilityinthesecriteria.However,thefollowinggeneralguidelinesshouldapplyinmostinstances.Ifthegoalissimplytokeepasmallbatchofpurifiedproteinaroundforafewdays,thenoftenstorageinunfrozensolutionat4℃issufficient.Ifgreaterdurationofstorageisrequiredorifthereisobviouslyproteindegradation(e.g.,visibleprecipitatesorunacceptablelossoffunction),thenthenextoptionistochooseeitherfrozenstorageorstorageasasalted-outprecipitate(UNIT4.5;APPENDIX3F).Bothmethodsarerelativelystraightforwardand,whenconductedproperly,shouldaffordseveralweeksormonthsofstoragestability.Priortoattemptingtodesignasaltingoutstrategy,preliminaryfreeze-thawingexperimentsshouldbeconductedtodetermineagivenproteinpreparation'sresistancetothisstress.Iftheproteinisstableduringthistreatment,thenlong-termstorageinthefrozenstatewillprobablyprovideadequatestability.However,ifpreliminaryexperimentsdocumentthattheproteinissensitivetoacutefreeze-thawingstress,itwillbenecessarytotesttheappropriatecryoprotectantsortoopttoexploretheutilityofsaltingouttheprotein.Thelatterisprobablysomewhateasier,butitdoesnottakealargeefforttooptimizefreeze-thawingresistance.Atthispoint,thechoiceofmethodtousedependsonpriorexperiencewiththeprotein,aswellasthehistoryofhowagivenlabhassuccessfullydealtwithproteinstoragestabilityissues.Finally,onlyifallothermethodsfailorifthereisaneedforverylong-termstorage(e.g.,18to24months)atambienttemperaturesshouldfreeze-dryingbeexplored.STORAGEASFREEZE-DRIEDSOLIDSAproperlyformulatedandfreeze-driedproteinpreparationcanremainstableforyears,evenatroomtemperature(CarpenterandChang,1996);however,developmentofoptimalsolutioncompositionsandprocessingconditionsforafreeze-driedformulationiswellbeyondthescopeofthetypicalacademiclab.Onemaindifficultyisthatthetypeoflyophilizersavailableinmostlabs,whichusuallyhavenocapacitytocontrolsampletemperature,cannotachievethelowlevelofresidualwater(e.g.,1%to2%bymass)thatisrequiredforlong-termstoragestabilityoffreeze-driedproducts.Todrysamplessufficiently,theirtemperaturemustbeincreasedtogreaterthanroomtemperatureduringtheterminalstagesofthefreeze-dryingprocess.Also,withouttemperaturecontrolduringdryingthesamplecancollapseintoaclump,whichmakesitevenmoredifficulttoremovewater.Evenifappropriateprocessingconditionscanbedevelopedwithagivenlyophilizer,itiscriticaltousethecorrectadditivestopreventproteindenaturationduringbothfreezinganddrying(reviewedinCarpenterandChang,1996;Carpenteretal.,1997).Thedisaccharidessucroseandtrehaloseareveryeffectiveatprotectingproteinsduringthesestressesandduringstorageinthedriedsolid.Thesesugarsarenonreducing.ReducingsugarssuchasglucoseandmaltoseshouldnotbeusedsincetheydegradeproteinsviatheMaillardreaction.Thedegreeofprotectionduringfreezingdependsontheinitialbulkconcentrationofsugarpresent,whereasthatduringdryingdependsonthesugar:proteinmassratio(CarpenterandChang,1996).Asnotedabove,freezingprotectionisduetopreferen-tialexclusionofthesugarfromtheprotein'ssurface,whichincreasesthefreeenergyofunfolding.Duringdrying,proteinmoleculesunfoldasthewaterhydratingtheirsurfaceisremoved.Sugarspreventthisdamagebyhydrogenbondingtothedriedproteininplaceofthelostwater.Thestoragestabilityofadriedformulationdependsonalowresidualwatercontent(e.g.,1%to2%bymass),retentionofthenativeproteinconformationduringfre-ezinganddrying(whichcanbeaccomplishedwithprotectiveadditives)andstorageofthesamplesbelowtheglasstransitiontemperatureoftheamorphousphase,whichcontainstheproteinandstabilizingsugars(CarpenterandChang,1996).Measuringthesephysicalparametersisoftenbeyondthecapabilitiesofmostacademiclabs.Thus,formostlabsfreeze-dryingshouldbeconsideredonlywhenallothermethodsforenhancingstoragestabilityhavebeenfoundtobeinadequate.Assumingthatsuboptimalprocessingwillbenecessary(i.e.,thereisnowaytocontrolsampletemperatureinthefreeze-drier)andthatcriticalphysicalparameterscannotbemeasured,isthereanychanceofobtainingastabledriedformulation?Theanswerismaybe.Thepracticalapproachistopreparetheproteinatarelativelyhighconcentration(whichincreasesresistancetofreezingandreducesvolumetobedried)withsufficienttrehaloseorsucrosetoinhibitunfoldingduringfreezinganddrying.Iftheproteinpreparationisresistanttofreezing,usuallyasugar:proteinmassratiointherangeof1to2isadequatetopreventproteinunfoldingduringdrying.Ifprotectionisrequiredduringfreezing,oftenaninitialsugarconcentrationof200to300mMisrequiredforoptimalprotection.Itmayalsobebeneficialtoincludeacarbohydratepolymer(e.g.,dextran,Ficoll,hydroxyethylstarch),whichcanprovidesamplebulkandincreasetheglasstransitiontemperatureoftheamorphousphase.Sometimesaddingabulkingagentisneededtopreventproteinfromescapingfromthecontainerduringdrying.Also,thesepolymericamorphousbulkingagentsmakeiteasiertodrythesampleswithoutcollapse.Anini-tialpolymerconcentrationof2%to3%(w/v)usuallyprovidesadequatebulking.Thesamplesareusuallyfrozenbye