Advanced genetic strategies for recombinant protei

JournalofBiotechnology115(2005)113–128AdvancedgeneticstrategiesforrecombinantproteinexpressioninEscherichiacoliHansPeterSørensen,KimKuskMortensen∗LaboratoryofBioDesign,DepartmentofMolecularBiology,AarhusUniversity,GustavWiedsVej10C,DK-8000AarhusC,DenmarkReceived29March2004;receivedinrevisedform26August2004;accepted30August2004AbstractPreparationsenrichedbyaspecificproteinarerarelyeasilyobtainedfromnaturalhostcells.Hence,recombinantproteinpro-ductionisfrequentlythesoleapplicableprocedure.Theribosomalmachinery,locatedinthecytoplasmisanoutstandingcatalystofrecombinantproteinbiosynthesis.Escherichiacolifacilitatesproteinexpressionbyitsrelativesimplicity,itsinexpensiveandfasthigh-densitycultivation,thewell-knowngeneticsandthelargenumberofcompatibletoolsavailableforbiotechnology.Especiallythevarietyofavailableplasmids,recombinantfusionpartnersandmutantstrainshaveadvancedthepossibilitieswithE.coli.Althoughoftensimpleforsolubleproteins,majorobstaclesareencounteredintheexpressionofmanyheterologousproteinsandproteinslackingrelevantinteractionpartnersintheE.colicytoplasm.HerewereviewthecurrentmostimportantstrategiesforrecombinantexpressioninE.coli.Issuesaddressedincludeexpressionsystemsingeneral,selectionofhoststrain,mRNAstability,codonbias,inclusionbodyformationandprevention,fusionproteintechnologyandsite-specificproteolysis,compartmentdirectedsecretionandfinallyco-overexpressiontechnology.Themacromolecularbackgroundforavarietyofobstaclesandgeneticstate-of-the-artsolutionsarepresented.©2004ElsevierB.V.Allrightsreserved.Keywords:Escherichiacoli;Recombinantproteinexpressionsystems;Inclusionbodies;Fusionproteins;RarecodontRNAs1.ThemodernrecombinantexpressionsystemAnumberofcentralelementsareessentialinthedesignofrecombinantexpressionsystems(Baneyx,1999;Jonassonetal.,2002).Expressionisnormallyinducedfromaplasmidharbouredbyasystemcom-patiblegeneticbackground.Thegeneticelementsof∗Correspondingauthor.Fax:[phone].E-mailaddress:[e-mail](K.K.Mortensen).theexpressionplasmidincludeoriginofreplication(ori),anantibioticresistancemarker,transcriptionalpromoters,translationinitiationregions(TIRs)aswellastranscriptionalandtranslationalterminators.1.1.TherepliconTherepliconofplasmidscontaintheoriginofrepli-cationandinsomecasesassociatedcisactingelements(delSolaretal.,1998).Mostplasmidvectorsusedinre-0168-1656/$–seefrontmatter©2004ElsevierB.V.Allrightsreserved.doi:10.1016/j.jbiotec.2004.08.004114H.P.Sørensen,K.K.Mortensen/JournalofBiotechnology115(2005)113–128combinantproteinexpressionreplicatebytheColE1orthep15Areplicon.Plasmidcopynumberiscontrolledbytheoriginofreplicationthatpreferablyreplicatesinarelaxedfashion(Baneyx,1999).TheColE1repli-conpresentinmodernexpressionplasmidsisderivedfromthepBR322(copynumber15–20)orthepUC(copynumber500–700)familyofplasmids,whereasthep15ArepliconisderivedfrompACYC184(copynumber10–12).Thesemulti-copyplasmidsarestablyreplicatedandmaintainedunderselectiveconditionsandplasmidfreedaughtercellsarerare(Summers,1998).Plasmidincompatibilityisdefinedastheinabil-ityoftwoplasmidstobestablymaintainedinthesamecell(Hardy,1987).Differentrepliconincompatibilitygroupsanddrugresistancemarkersarerequiredwhenmultipleplasmidsareemployedfortheco-expressionofgeneproducts.DerivativescontainingColE1andp15Arepliconsareoftencombinedinthiscontextsincetheyarecompatibleplasmids(Mayer,1995).1.2.ResistancemarkersThemostcommondrugresistancemarkersinre-combinantexpressionplasmidsconferresistancetoampicillin,kanamycin,chloramphenicolortetracy-cline.Plasmidmediatedresistancetoampicillinisac-complishedbyexpressionof
-lactamasefromtheblagene.Thisenzymeissecretedtotheperiplasm,whereitcatalysehydrolysisofthe
-lactamring.Ampicillinpresentinthecultivationmediumisespeciallysuscep-tibletodegradation,eitherbysecreted
-lactamase,oracidicconditionsinhigh-densitycultures.Thelattereffectcanbealleviatedbythelessdegradationsus-ceptibleampicillinanalog,carbenicillin,Kanamycin,chloramphenicolandtetracyclineinterferewithpro-teinsynthesisbybindingtocriticalareasoftheri-bosome.Kanamycinisinactivatedintheperiplasmbyaminoglycosidephosphotransferasesandchloram-phenicolbythecatgeneproduct,chloramphenicolacetyltransferase.Variousgenesconferresistancetotetracycline(Connelletal.,2003).1.3.PromotersRecombinantexpressionplasmidsrequireastrongtranscriptionalpromotertocontrolhigh-levelgeneexpression.Basaltranscriptionintheabsenceofinducerisminimizedthroughthepresenceofasuitablerepressor.Minimizationofbasaltranscriptionisespeciallyimportantwhentheexpressiontargetintroduceacellularstresssituationandtherebyselectsforplasmidloss.Promoterinductioniseitherthermalorchemicalandthemostcommoninduceristhesugarmoleculeisopropyl-beta-d-thiogalactopyranoside(IPTG)(HannigandMakrides,1998).1.4.MessengerRNATranslationinitiationfromthetranslationinitiationregion(TIR)ofthetranscribedmessengerRNAre-quirearibosomalbindingsite(RBS)includingtheShine–Dalgarno(SD)sequenceandatranslationinitia-tioncodon(Sørensenetal.,2002).TheShine–Dalgarnosequenceislocated7±2nucleotidesupstreamfromtheinitia