蛋白质纯化的原理和方法Word格式.docx
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蛋白质纯化的原理和方法Word格式.docx
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•Celldisruption:
Periplasmicandcytoplasmicproteinsarereleased
•Centrifugationleadstoasolublefraction(supernatant)whichcontainsallsolubleperiplasmicandcytoplasmicproteinsandamembranefractionfromwhichmembraneboundproteinscanbesolubilisedwithdetergents(e.g.TritonX-100)
•Thesolubleormembranefractionarethestartpointofthefurtherpurificationbychromatography
Celldisruption:
FrenchPress
Lysozyme
Ultrasonic
MembraneProteins
•Peripheralmembraneproteins:
inmostcasessolubleinbufferswithhighorlowionicstrengthorhighpH
•Integralmembraneproteins:
theycontaintransmembranehelicesandmustbesolubilisedtoconserveconformationandfunctionoftheprotein
Solubilisationofintegralmembraneproteins
•SolubilisationofproteinsisdonewithadetergentsconcentrationabovetheCMCtoensuretheincorporationofmembranelipidintodetergentmicelles.
•CMC=criticalmicelleconcentration
dependsontemperature,ionicstrengthandpHofthebufferandconcentrationofunchargedsubstanceslikeureaoralcohol
Somedetergents
•Ionicdetergents:
Sodium-Dodecylsulfate:
denaturesProteins(
SDS-PAGE)
Na-Deoxycholate:
preticipatesbypH<
6.8
preticipatewithCa2+orMg2+
InGeneral:
NoionicDetergentsinpurificationswithdependonthechargeoftheproteins.
•Non-ionicdetergents:
TritonX-100
Phenylgroups
strongAbsorbanceat280nm
Tween20
likeTritonX-100notdialyzable
•Zwitter-ionicdetergents
Chaps
dialyzable
Whichproteinsarepurified?
•Metabolicpathways
•Energyproduction
Aim:
biochemicalcharacterisation(Reactivity,subunitcomposition,organicandinorganiccofactors,3Dstructure)
…andwhy?
Purificationstrategies
•Proteinstabilisation:
–IntegralmembraneProteins:
Solubilisation
–Purificationat4°
C:
reducesproteaseactivity
–Additionofproteaseinhibitors:
commonlyusedareEDTAandPMSF(toxic)
–Quicklyloadonfirstcolumnaftercelldisruptionandultracentrifuge
•Mainimpuritiesareremovedfirst,lesserinthesecondorthirdstep
•Max5%impuritiesareacceptable
Ingeneral:
Max4purificationsteps
Nostepswithpurificationfactor<
5
Nostepswith<
30%yield
Nostepswhichlastlongerthanonedayandonenight
Chromatography
•Separationmaterialincolumns,streamedbybuffer
→liquidchromatography
HPLC:
highpressure/performanceliquidchromatography
FPLC:
fastproteinliquidchromatography
FPLCunit
Separationprinciples
•Size:
sizeexclusionchromatography(=gelfiltration,=gelpermeationchromatography)
•Charge
anionorcationexchangechromatography;
chromatofocusing
•Hydrophobicity
hydrophobicinteractionchromatography(HIC)
•Affinity
affinitychromatography
•Solubility
ammoniumsulfateprecipitation(nonchromatographic,rel.imprecise)
Sizeexclusionchromatography
•Differentporesizes,dependingonthesizeoftheproteins
•Separationisbasedindiffusion
slowflowrate
•Pressuresensitivematerials
Exampleforaseparationbygelfiltration
Noteworthyaboutchromatography
Gelfiltration:
limitedsamplevolume:
2-3mllimitedflowrate
gelfiltraiontakestimedelutionofthesamplebyafactorofabout3lowpurificatopnfactor:
3-6needscolumnbufferwithhighionicstrength:
min0.1M
Ionexchagechromatography:
Toremember:
Proteinbindingdepentsonelectrostaticinteractionswiththecolumnmaterial.StrengthofbindingdepentsonpHandionicstrengthofthebuffer,thepIoftheproteinandthechargedensityonthecolumn.
Technicaleasierthangelfiltration:
columnscouldbepackedattheFPLC.Samplevolumecanbemultipletimesthecolumnvolume.Higherflowrates.Purificationfactor:
3-15Samplegetsconcentrated.
Don’tusechargeddetergents!
Ionexchagechromatography
Bindingbehaviourofproteins
•pI=isoelectricpointoftheprotein
thepHvalueatwhichthepossesnonetcharge
•ThepIdeterminesthechargeoftheproteinatagivenpH
pH>
pI
negativenetcharge
pH<
positivenetcharge
pIandproteinseparationonionexchangecolumns
But...
•pIisnotaloneresponsibleforthebindingbehaviourofproteins
Bindingissometimesinfluencedbylocalchargesandnotbythenetchargeoftheprotein
Exampleforaseparationbyionexchangechromatography
Materials
Carriermaterials
•Polysugars
–Sepharose(Agarose),Sephadex(Dextran),Sephacel(Cellulose)
–Raresugarscanhardlybeutilisedbybacteria.
–Lowflowrates
–Materialchangesit’sVolumedependingontheionicstrangth
–Materialsettlesovertime
•Beads
–Polystyrol/Divinylbenzenbeadswithchargecarrieradded
–Relativehighflowrates–Goodpressurestability,nocompression
–Hardchargescouldstresstheprotein
•Beadsandtentacle
–Chargesarelinkedwithflexiblespacerstopolymericbeads.Thisleadstoasoftbindingofproteinsdespiteofhardchargesonthematrix
–Relativehighflowrates
–Goodpressurestability
–Highcapaticity
•Perfusionbeads
–Porousmaterial,beadswithchanels
–Verybigsurface
–Highlypressurestable
–Veryhighflowrates
Qualityoftheseparation
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- 蛋白质 纯化 原理 方法
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