Kevin Ahern's Biochemistry Course (BB 350) at Oregon State University
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ProteinStructure&Puri ication
1.Disruptionofforcesthatstabilizeproteinstructurecausefoldedproteinstounfold.Unfolded
proteinsarenotfunctional.Wedescribethemasdenatured.Denaturingagentsincludeheat,detergent,
acidorbase.
2.RNaseisanunusualenzyme.Itisverystable.Afterbeingheatedtoboilingtemperatures,itwill
renatureandregainitsactivity.Mostproteinsdon'tdothis.
3.Foldingofaproteinishowitgetsits3Dshape.Foldingisaprocessthatisdeterminedbythe
sequenceofaminoacidsinaproteins.
4.Misfoldedproteinscauseenormousproblemssometimes.Amisfoldedproteinisthecauseofmad
cowdisease.Themisfoldedproteiniscalledaprionanditworksbyinducingothercopiesofthesame
proteintomisfold.
5.Chaperoninsarecomplexesthatfacilitatetheproperfoldingofproteins.
6.Tostudyaprotein,onemustpurifyitawayfromalltheotherproteinsinacell.Stepsinthe
puri icationtypicallyinclude1)bustingcellsopen;2)centrifugingcellularcomponentsapartfromthe
cytoplasm;and3)usingtechniquesthatseparatemoleculesbyseveraldifferentprocesses.
7.Mostproteinsrequiremorethanonemethodtobepuri ied.
8.Techniquesforproteinpuri icationoftenrelyonthestructuralcomponentsofproteins.Isolationof
proteinsfromcellsrequiresbreakingopenthecells,centrifugationtoremoveinsolubledebris,andthe
applicationofproteinisolationtechniquestopurifydesiredproteinsfromthesolublefractionofthe
cells.
9.Gelexclusionchromatographyisatechniqueforisolatingproteinsonthebasisoftheirdifferent
sizes.Themethoduses'beads'withuniformholesinthem.Theholesareopeningstotunnelsthrough
thebead.Smallmoleculesthat itintotheholestravelthroughtthetunnelsandtakelongertopass
throughthecolumnthanlargemoleculesthatdonot itintotheholes.
A9 10.Af initychromatographyusesthestructureofamoleculethataproteinbinds(suchasATP)asa
meansofpurifyingtheprotein.Forexample,proteinsthatbindATPwouldberetainedbyacolumnfull
ofbeadswithATPontheirsurface.Thenon-ATP-bindingproteinswillpassthrough irst.ATP-bindingproteinscanberemovedfromthecolumnbyaddingATP.
11.Ionexchangechromatographyusesionicinteractionsasameansofseparatingproteins.Cation
exchangechromatographyusesnegativelychargedbeads.Theseattractthepositivelychargedproteins
inamixture,whereasthenegativelychargedproteinspassthroughquickly.Anionexchange
chromatographyhasanoppositestrategy.
12.Gelelectrophoresisinvolvesgelatinoussupportmaterials(agarose-forDNAorpolyacrylamideusuallyforproteins)andanelectriccurrentthatdragsmoleculesthroughthegel.Electrodesare
arrangedsuchthatthe"top"orbeginningofthegeliswherethenegativeelectrodeisplacedandthe
positiveelectrodeisplacedatthebottomorendofthegel.DNAisnegativelycharged,soitisrepelled
awayfromthetopandtowardsthebottomofthegel.Separationisonthebasisofsize.Largemolecules
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Kevin Ahern's Biochemistry Course (BB 350) at Oregon State University
2 of 2
http://oregonstate.edu/instruct/bb350/spring13/highlightsprotpurif.html
travelslowestinthegel,whereasthesmallmoleculestravelfastest.DNAfragmentsappearasbandson
agelandbandscanbeexcisedseparatelyfromtheotherbandsforfurthermanipulation.
13.Proteinscanhavenegative,positive,ornocharge.Toworkinelectrophoresis,theymustbe
convertedfromfoldedentitiesintorodswithuniformnegativecharge.Thisisdoneusingthedetergent
calledSDS.Itunfoldstheproteinandcoatsitwithnegativecharges.Thiscoatedproteinisloadedonto
agelofpolyacrylamideandthecomplexesaresortedonthebasisofsize,justliketheDNAfragments
were-smallestmovesfastestandlargestmovesslowest.
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