Asuccessfulproteinpurificationprocedurecanbenothingshortofamazing.WhetheryouarestartingoffwitharecombinantproteinwhichisproducedinE.coli,ortryingtoisolateaproteinfromsomemammaliantissue,youaretypicallystartingwithgramquantitiesofacomplexmixtureofprotein,nucleicacids,polysaccharide,etc.fromwhichyoumayhavetoextractmilligram(ormicrogram!)quantitiesofdesiredproteinathighpurity,andhopefullywithhighyield.

Thespecificassaycanbebaseduponsomeuniquecharacteristicoftheproteinofinterest

• Enzymaticactivity
• Immunologicalactivity
• Physicalcharacteristics(e.g.molecularmass,spectroscopicproperties,etc.)
• BIOLOGicalactivity
• Ideally,anassayshouldbe
  • Specific
  (youdon\"twantafalsepositive)
  • rapid
  (youdon\"twanttowaitaweekfortheresults)
  • sensitive
  (youdon\"twanttoconsumeallyoursampleinordertoassayit)
  • quantitative
  (youneedanaccuratewaytomeasurethequantityofyourproteinateachstepinthepurification)

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AntibodiescanbeusedinamethodcalledWesternblotting,whichisusefulfordetermininglevelsofproteinexpressionandforassayingproteinsduringpurification.Thismethodusuallyinvolvesthefollowingsteps:

• Aproteinsampleissubjectedtopolyacrylamidegelelectrophoresis.
• Afterthisthegelisplacedoverasheetofnitrocelluloseandtheproteininthegeliselectrophoreticallytransferedtothenitrocellulose.
• Thenitrocelluloseisthensoakedingelatinto\"block\"itsABIlitytonon-specificallybindproteins.
• Thenitrocelluloseisthenincubatedwiththespecificantibodyfortheproteinofinterest.
• Thenitrocelluloseisthenincubatedwithasecondantibodywhichisspecificforthefirstantibody.Forexample,ifthefirstantibodywasraisedinrabbits,thesecondantibodymightbetermed\"goatanti-rabbitimmunoglobulin\".Whatthismeansisthatrabbitimmunoglobulinswereusedtoelicitanantibodyresponseingoats.Thegoatantibodies(polyclonal)willincludethosewhichrecognizetheconservedregionintherabbitantibodies.SincetheFcregionisconserved,itwillbindtoanyandallrabbitantibodies,includingthoseonthenitrocellulosepaper.
• Thesecondantibodywilltypicallyhaveacovalentlyattachedenzymewhich,whenprovidedwithachromogenicsubstrate,willcauseacolorreaction.
• Thusthemolecularweightandamountofthedesiredproteincanbecharacterizedfromacomplexmixture(e.g.crudecellextract)ofotherproteins.

Inavariationoftheabove,theproteinsamplemaybeblotteddirectlyonanitrocellulosepaper(calledadotblot)withoutfirstrunningagel.Thismaybedesirableif,forexample,theantibodyismonoclonalandrecognizesanepitopewhichisdependentuponnativestructure(whichwouldbedestroyeduponrunninganSDSPAGE).

Inadditiontotheirvarieduses,antibodiescanalsobeusedtopurifyproteins.

• Ifrelativelylargeamountsofanantibodycanbeobtained,theycanbecovalentlyattachedtoachromatographyresin(e.g.sephadexbeads).
• Ifacrudecellextractisrunoversuchacolumn,onlytheproteinofinterestshouldbind,andeverythingelsewillflowthrough.
• Theboundproteincanthenbeeluted.ThisistypicallyachievedbymoderatelylowpHconditions(usingaceticacid).AslongastheproteinofinterestisnotirreversIBLydenaturedbysuchconditions,themethodwillworkquitewell.
• Onepotentialpitfallinvolvesthatofmonoclonalantibodiesbeingutilizedtopurifymutantproteins.Theregionsoftheproteincomprisingtheepitopecannotbemodifiedwithoutdestroyingtheabilityoftheantibodytobind.Thus,theuseofmonoclonalantibodiesinapurificationschememayprecludeitsuseinpurifyingcertainmutants.

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Proteinpurificationcanbethoughtofasaseriesoffractionationstepsdesignedsothat:

• Theproteinofinterestisfoundalmostexclusivelyinonefraction(andwithgoodyield)
• Asignificantamountofthecontaminantscanbefoundinadifferentfraction

Duringpurificationyouwillneedtomonitorseveralparameters,including:

• Totalsamplevolume
• Totalsampleprotein
(canbeestimatedbyA₂₈₀;1.4~1.0mg/ml)
• Unitsofactivityofdesiredprotein
(basedonspecificassay)

Thisbasicinformationwillallowyoutokeeptrackofthefollowinginformationduringeachstepofpurification:

• %yield
foreachpurificationstep
• Specificactivity
ofthedesiredprotein(units/mgtotalprotein)
• Purificationenhancement
ofeachstep(e.g.\"3.5xpurification)

Indesigningapurificationschemeyoutypicallyhavetobalancepurificationwithyield.

• Forexample,itmayberelativelystraightforwardtoobtain90%purematerialwithgoodyield.
• However,itmaybedifficulttoimprovethatpurityanadditionalfewpercentilewithgoodyield.
• Theplannedapplicationofthepurifiedproteindeterminesthetargetpurity
.
• Iftheproteinistobeusedtodetermineaminoacidsequenceinformation,maybe90%isacceptable.However,ifthematerialistobeusedinclinicaltrials,99.99+%maybethetargetpurity.

Initialstepsinpurification



• Itisextremelyhelpfultohavesomeinformationnotonlyonthegeneralphysicalandchemicalcharacteristicsoftheproteinyouaretryingtopurify,butalsoonthecontaminatingcomponents.
• Forexample,manyE.coliproteinsaregenerallylowmolecularweight(<50,000Da)andsomewhatacidicinisoelectricpoint

Usuallytheinitialstepsinpurificationmakeuseofgeneralphysicaland/orchemicaldifferencesbetweensolubleproteinsandothercellcomponents.

• Forexample,solubleproteinscanbeseparatedfromgeneralcellulardebris,andintactcells,bycentrifugation.
• Thus,cellsarephysicallydisrupted(viahomogenizationoracellpress)toallowreleaseofcellcontents.Thisisthenfollowedbycentrifugationtoseparategenerallysolublecomponentsfromthosewhichareinsoluble.
• Itisatthispointthatdatacollectionbeginsinordertomonitorthepurification.

NucleicacidscansometimesbereADIlyremovedfromthesamplebytheadditionoflargecationiccompoundssuchaspolyethyleneimine,orstreptomycinsulfate.

• Thenucleicacidsbindtothesecompoundsviaelectrostaticinteractionsandthecomplexprecipitatesandcanberemovedviacentrifugation.
• Thesamegeneralresultcanbeobtainedbymixinginionexchangeresinswhichareanionexchangers(i.e.theresinscontaincationicgroups)andthenfilteringorcentrifugingtoremove.Aswitheithermethod,itshouldbeconfirmedthatthedesiredproteinisnotboundaswell.

Crudefractionationsofproteinscanbeachievedbyaddingvariousquantititesofprecipitantssuchasammoniumsulfate,orpolyethyleneglycol(PEG).

• Forthistypeofpurificationstepaninitialexperimentisperformedtomonitorthefractionofoverallprotein,aswellasdesiredprotein,remaininginsolution(andpellet)asafunctionofprecipitantconcentration.

AmmoniumSulfate(%saturated)	0	10	20	30	40	50	60	70	80	90
SampleA280	1000	900	600	200	100	75	50	40	25	20
Activityassay(units)	200	200	200	190	170	100	30	5	0	0 Inthisparticularexampleweareinluck:ataround30%ammoniumsulfatewecanprecipitateabout80%ofthetotalproteinconcentrationinoursample,yetouractivityassayforourdesiredproteinindicatesthatabout95%ofourdesiredproteinisstillsoluble. At80%ammoniumsulfateallofourdesiredproteinhasprecipitated.Thus,fromtheseresultswewoulddothefollowing: Addammoniumsulfatetooursampletoaconcentrationof30%saturation Centrifugeanddiscardthepellet Addammoniumsulfateto80%saturation Centrifugeandkeepthepellet.ResUSPendthepelletinbuffertosolubilizetheprotein. Wewouldexpectabouta5-foldpurificationwithabout95%yield.