Efficient site directed in vitro mutagenesis using ampicillin selection potx

... NucleicAcidsResearch,Vol.18,No.123439 Efficient site directed in vitro mutagenesis using ampicillin selection MartinK.Lewis*andDavidV.ThompsonPromegaCorporation,Madison,WI53711,USAReceivedApril11,1990;AcceptedMay25,1990ABSTRACTAnovelplasmidvectorpSELECT-1isdescribedwhichcanbeusedforhighly efficient site- directed in vitro mutagenesis. The mutagenesis methodisbasedontheuseofsingle-strandedDNAandtwoprimers,onemutagenicprimerandasecondcorrectionprimerwhichcorrectsadefect in the ampicillin resistancegeneonthevectorandrevertsthevectorto ampicillin resistance. Using T4DNApolymeraseandT4DNAligasethetwoprimersarephysicallylinkedonthetemplate.Thenon-mutantDNAstrandisselectedagainstbygrowth in thepresenceof ampicillin. In testsofthevector,highly efficient (60-90%) mutagenesis wasobtained.INTRODUCTION Site- directed in vitro mutagenesis isavaluabletechniqueforamongotherthingsthestudyofcriticalaminoacidresiduesinvolved in enzymaticactivity,thestudyofDNApromoterandenhancerfunctionandstructure,thestudyofresiduesimportant in proteinfolding,thestudyofthestructureofDNAbindingsitesforproteins,thestudyoffunctionsofparticularresiduesordomains in proteinstability,thecreationofmutantproteinswithincreasedstabilityorresistancetoenvironmentalagents,thestudyofeffectsofremovingsitesforproteinmodification,suchasphosphorylationorglycosylationandforengineeringofexpressionclones.Hutchisonetal.(1)introducedageneralmethodtoobtain site- specificchanges in DNAsequences using single-strandedDNA(ssDNA)andasyntheticoligonucleotide.Theoligonucleotideiscomplementarytothesingle-strandedtemplateDNAexceptforaregionofmismatch in thecenter.Followinghybridization,theoligonucleotideisextendedwithDNApolymerasetocreateadouble-strandstructure.ThenickissealedandtheresultingheteroduplexistransformedintoanE.colihost.UponDNAreplicationandstrandsegregation,thecellcontainsamixtureofwild-typeandmutanttemplates.Becausemutantandwild-typeplasmidsarepresent in thesamecell,asecondroundoftransformationisgenerallyemployedtoensuregeneticpurity.Thoughtheoreticallytheyieldofmutants in theaboveprocedureshouldbe50% in practice,itisgenerallymuchlower,oftenonlyafewpercent.Various selection techniqueshavebeenemployedtoincreasetheefficiencyof site- directed in vitro mutagenesis (2,3).Wedescribeanovelphagemidvectorand selection techniquewhichresults in ahighproportion(60-90%)ofmutants.MATERIALSANDMETHODSMaterialsAllrestrictionenzymesandDNAmodifyingenzymeswereobtainedfromPromegaCorporation.OligonucleotidesweresynthesizedonanAppliedBiosystems380BDNAsynthesizer using phosphoramiditechemistry.Allchemicalswereofreagentgrade.ConstructionofpSELECT-1pSELECT-lisacloningvectorspecificallyconstructedforuse in in vitro mutagenesis. ThevectorisahybridoftheplasmidspBR322(4,5)andpGEM-3Zf(+)(PromegaCorporation,Madison,Wisconsin).Thevectorcarriesmodified ampicillin andtetracyclineresistancegenesderivedfrompBR322and in additioncarriesthepolylinkerandflreplicationoriginfrompGEM-3Zf(+).ToconstructpSELECT-1(seeFigure1),the ampicillin resistancegeneofpBR322wasinactivatedbydigestingtheDNAwithPstI,bluntingtheends using theKlenowfragmentofDNApolymeraseIandrecircularizingthevector using T4DNAligase.Thisintroducedafour-baseframeshiftwhichwascheckedbyDNAsequencingandwasfoundtomakethevector ampicillin sensitive.LigationmixesweretransformedintoE.coliJM109andplatedonLBplatescontaining15,tg/mltetracycline.ToclonethesegmentsofpGEM-3Zf(+)intothismodifiedpBR322,theformerwasdigestedwithAatIIandAflIIIandthelatterwithAatIIandEcoR1.Thedigestsweremixedtogetherandligatedfortwohours,allowingtheAatIIendofthepGEM-3Zf(+)fragmenttoligatetotheAatIIendofthemodifiedpBR322.TheDNAendswerethenbluntedbyfilling in withKlenowandtheligationthenallowedtoproceedovernight.ThisstepallowstherecircularizationoftherecombinantplasmidbybluntendligationofthefilledAflIIIandEcoRIends.TheligationmixwasplatedonLBplatescontainingtetracycline,IPTGandX-Galandscoredfortetracyclineresistant*Towhomcorrespondenceshouldbeaddressed.::)1990OxfordUniversityPress3440NucleicAcidsResearch,Vol.18,No.12bluecolonies.ToobtainacolonywhichisbothtetracyclineresistantandbluewouldindicatethesuccessfulcloningofthepGEM-3Zf(+)AatII-AflIIIfragment(whichcarriesthelacalphapeptideandhenceconfersbluecolortoJM109)intothetetracyclineresistantmodifiedpBR322betweentheAatIIandEcoRIsites.AbluetetracyclineresistantcolonywasfoundandthestructureoftheresidentplasmidwascheckedandfoundtobethecorrectfragmentinsertedintothemodifiedpBR322.ThisplasmidwasnamedpBR322ZF.ItwaspredictedthattheEcoRI site shouldhavebeenreformedattheAflHI-EcoRIjunction,and in factrestrictionmappingindicatedthatthiswasthecase.ThoughtheconstructnowcontainedthepGEM-3Zf(+)polylinker,manyofthesesiteswerenolongerunique. In particular,theHindII,BamH1,SphIandSalIsites in thelinkerwerealsopresent in thetetracyclineresistance(tet)gene. In ordertoremovethesesitesfromthetetgene,anotherderivativeofpBR322wasconstructed. In thiscaseonlytheFloriginregionfrompGEM-3Zf(+)wasclonedintothe ampicillin sensitivepBR322derivativeonanAatIl-EcoRIfragmentbetweentheAatIIandEcoRIsitesonthisvector.This ... NucleicAcidsResearch,Vol.18,No.123439 Efficient site directed in vitro mutagenesis using ampicillin selection MartinK.Lewis*andDavidV.ThompsonPromegaCorporation,Madison,WI53711,USAReceivedApril11,1990;AcceptedMay25,1990ABSTRACTAnovelplasmidvectorpSELECT-1isdescribedwhichcanbeusedforhighly efficient site- directed in vitro mutagenesis. The mutagenesis methodisbasedontheuseofsingle-strandedDNAandtwoprimers,onemutagenicprimerandasecondcorrectionprimerwhichcorrectsadefect in the ampicillin resistancegeneonthevectorandrevertsthevectorto ampicillin resistance. Using T4DNApolymeraseandT4DNAligasethetwoprimersarephysicallylinkedonthetemplate.Thenon-mutantDNAstrandisselectedagainstbygrowth in thepresenceof ampicillin. In testsofthevector,highly efficient (60-90%) mutagenesis wasobtained.INTRODUCTION Site- directed in vitro mutagenesis isavaluabletechniqueforamongotherthingsthestudyofcriticalaminoacidresiduesinvolved in enzymaticactivity,thestudyofDNApromoterandenhancerfunctionandstructure,thestudyofresiduesimportant in proteinfolding,thestudyofthestructureofDNAbindingsitesforproteins,thestudyoffunctionsofparticularresiduesordomains in proteinstability,thecreationofmutantproteinswithincreasedstabilityorresistancetoenvironmentalagents,thestudyofeffectsofremovingsitesforproteinmodification,suchasphosphorylationorglycosylationandforengineeringofexpressionclones.Hutchisonetal.(1)introducedageneralmethodtoobtain site- specificchanges in DNAsequences using single-strandedDNA(ssDNA)andasyntheticoligonucleotide.Theoligonucleotideiscomplementarytothesingle-strandedtemplateDNAexceptforaregionofmismatch in thecenter.Followinghybridization,theoligonucleotideisextendedwithDNApolymerasetocreateadouble-strandstructure.ThenickissealedandtheresultingheteroduplexistransformedintoanE.colihost.UponDNAreplicationandstrandsegregation,thecellcontainsamixtureofwild-typeandmutanttemplates.Becausemutantandwild-typeplasmidsarepresent in thesamecell,asecondroundoftransformationisgenerallyemployedtoensuregeneticpurity.Thoughtheoreticallytheyieldofmutants in theaboveprocedureshouldbe50% in practice,itisgenerallymuchlower,oftenonlyafewpercent.Various selection techniqueshavebeenemployedtoincreasetheefficiencyof site- directed in vitro mutagenesis (2,3).Wedescribeanovelphagemidvectorand selection techniquewhichresults in ahighproportion(60-90%)ofmutants.MATERIALSANDMETHODSMaterialsAllrestrictionenzymesandDNAmodifyingenzymeswereobtainedfromPromegaCorporation.OligonucleotidesweresynthesizedonanAppliedBiosystems380BDNAsynthesizer using phosphoramiditechemistry.Allchemicalswereofreagentgrade.ConstructionofpSELECT-1pSELECT-lisacloningvectorspecificallyconstructedforuse in in vitro mutagenesis. ThevectorisahybridoftheplasmidspBR322(4,5)andpGEM-3Zf(+)(PromegaCorporation,Madison,Wisconsin).Thevectorcarriesmodified ampicillin andtetracyclineresistancegenesderivedfrompBR322and in additioncarriesthepolylinkerandflreplicationoriginfrompGEM-3Zf(+).ToconstructpSELECT-1(seeFigure1),the ampicillin resistancegeneofpBR322wasinactivatedbydigestingtheDNAwithPstI,bluntingtheends using theKlenowfragmentofDNApolymeraseIandrecircularizingthevector using T4DNAligase.Thisintroducedafour-baseframeshiftwhichwascheckedbyDNAsequencingandwasfoundtomakethevector ampicillin sensitive.LigationmixesweretransformedintoE.coliJM109andplatedonLBplatescontaining15,tg/mltetracycline.ToclonethesegmentsofpGEM-3Zf(+)intothismodifiedpBR322,theformerwasdigestedwithAatIIandAflIIIandthelatterwithAatIIandEcoR1.Thedigestsweremixedtogetherandligatedfortwohours,allowingtheAatIIendofthepGEM-3Zf(+)fragmenttoligatetotheAatIIendofthemodifiedpBR322.TheDNAendswerethenbluntedbyfilling in withKlenowandtheligationthenallowedtoproceedovernight.ThisstepallowstherecircularizationoftherecombinantplasmidbybluntendligationofthefilledAflIIIandEcoRIends.TheligationmixwasplatedonLBplatescontainingtetracycline,IPTGandX-Galandscoredfortetracyclineresistant*Towhomcorrespondenceshouldbeaddressed.::)1990OxfordUniversityPress3440NucleicAcidsResearch,Vol.18,No.12bluecolonies.ToobtainacolonywhichisbothtetracyclineresistantandbluewouldindicatethesuccessfulcloningofthepGEM-3Zf(+)AatII-AflIIIfragment(whichcarriesthelacalphapeptideandhenceconfersbluecolortoJM109)intothetetracyclineresistantmodifiedpBR322betweentheAatIIandEcoRIsites.AbluetetracyclineresistantcolonywasfoundandthestructureoftheresidentplasmidwascheckedandfoundtobethecorrectfragmentinsertedintothemodifiedpBR322.ThisplasmidwasnamedpBR322ZF.ItwaspredictedthattheEcoRI site shouldhavebeenreformedattheAflHI-EcoRIjunction,and in factrestrictionmappingindicatedthatthiswasthecase.ThoughtheconstructnowcontainedthepGEM-3Zf(+)polylinker,manyofthesesiteswerenolongerunique. In particular,theHindII,BamH1,SphIandSalIsites in thelinkerwerealsopresent in thetetracyclineresistance(tet)gene. In ordertoremovethesesitesfromthetetgene,anotherderivativeofpBR322wasconstructed. In thiscaseonlytheFloriginregionfrompGEM-3Zf(+)wasclonedintothe ampicillin sensitivepBR322derivativeonanAatIl-EcoRIfragmentbetweentheAatIIandEcoRIsitesonthisvector.This ... allowedonetomakesingle-strandedDNA(ssDNA)containingthetetgeneandhencemodifythisgeneby site- specific in vitro mutagenesis. ThisvectorwasnamedpBR322F1.Single-strandedDNAwasmadefromthisvectorbypropagatingtheplasmid in E.coliNM522andinfectingwithM13K07helperphage. In vitro mutagenesis toremovetheHindIm site wasperformedbyhybridizinganoligonucleotidehavingthesequencepGCTTATCATCGATTA-GC'Tl'l'AATGCGGtothessDNA.ThisoligonucleotideremovestheHindIII site present in thetetracyclineresistancegenepromoterbychangingthefirstA in thesequenceAAGCTTtoaT.About0.1gofsingle-strandedtemplatewasusedandanoligonucleotide:vectorratioofabout15.Thehybridizationconditionswere25mMTris-HClpH7.3,12mMMgCl2and60mMNaCl in avolumeof25Al.Theannealingreactionwasheatedto700Cfor5minutesandthencooledtoroomtemperatureoverthecourseof15minutes.Thenallfourdeoxyribonucleotides(dATP,dCTP,dGTP,dTTP)wereaddedtothereactiontoafinalconcentrationof1mM,10unitsofT4DNApolymeraseand2unitsofT4DNAligase.Theseadditionsincreasedthereactionvolumeto35Al.Thefill in reactionwasallowedtoproceedfor90minutesat37°CatwhichpointtheentirereactionwastransformedintocompetentBMH71-18mutSE.coliandthetransformationmixtureaddedtoa50mlLBculturecontaining15Ag/mltetracyclineandtheculturegrownupovernight.PlasmidDNAwasthenpreparedfromthisculture using amini-prepprocedure,theDNAwasrestrictedwithHindIII(toselectforthosemutantsmissingtheHindIII site) ,transformedintoE.coliJM109andthecellsplatedonLBplatescontaining15pg/mltetracycline.TwotetracyclineresistantcolonieswereisolatedandplasmidDNApreparedfromtheseisolates.Restrictionenzymedigestionindicatedthatbothisolateshad in factdeletedtheHindHI site. TodeletetheBamH1,SphIandSalIsitesfromthetetracyclineresistancegene,oligonucleotidesweredesignedwhichremovedeachrestriction site whilekeepingtheaminoacidsequenceofthetetproteinunchanged.TherespectiveoligonucleotidesusedwerepCCCGTCCTGTGGATTCTCTA-CGCCGG,pGGCGCCATCTCCTTACATGCACCATTCCT-TGCGandpTCGCATAAGGGAGAGCGCCGACCCATGC-CCTTG. In eachcasethe mutagenesis procedurewasfollowedessentiallyasaboveandbasicallyinvolvedahybridization,an in vitro fill in, atransformation,aplasmidpreparation,arestrictionenzymerecutandaretransformation.Thiscompletedtheengineeringofthetetracyclineresistancegenesothatitwouldbeusefulwhenincorporatedintothe mutagenesis vector.TotransferthemodifiedtetgeneintopBR322ZF,thegenewasexcisedonaClaI-StyIfragment,gelpurifiedandclonedintopBR322ZFbetweentheClaIandStyIsites.Next,oneofthetwoEcoRIsites in theresultingvectorwasremoved.The site removedwastheoneoutsidethepolylinkeranditwasdestroyedbypartialEcoRIdigestion,fillingwithKlenowandreligating,followedbyrestrictionenzymedigestiontomapwhich site wasremovedfromisolateswhichcutonlyoncewithEcoRI.TheresultingvectorwasnamedpSELECT-1.RESULTSReversionofpSELECT-1to Ampicillin ResistancepSELECT-lisaplasmidspeciallyengineeredforusein...

... Combining Site- Specific Chemical Modification with Site- Directed Mutagenesis: Versatile Strategy to Move Beyond StructuralLimitations of 20 Natural Amino Acids Side Chains in Protein EngineeringGrace ... Deletions, and Insertions, Using QuikChange™ Site- Directed Mutagenesis Wenyan Wang and Bruce A. Malcolm 376 Efficient and Accurate Site- Directed Mutagenesis of Large PlasmidsSusan A. Nadin-Davis ... Braman © Humana Press Inc., Totowa, NJ2 Site- Directed Mutagenesis Using Alteredβ-Lactamase SpecificityChristine A. Andrews and Scott A. Lesley1. Introduction Site- directed mutagenesis (SDM) is...

... Using the Unique Restriction Site Elimination (USE) Method Li Zhu 1. Introduction In vitro site- directed mutagenesis has been widely used in vector modi- fication, and in gene and protein ... restriction site located in an intergemc region as the selection site. If the selection restriction site must be located within a gene, avoid using a selection primer that will introduce changes ... Acad Scl. USA 82,488492. Mutagenesis Using the USE Method 6. Lewis, M. K and Thompson, D. V. (1990) Efficient site- directed in vitro mutagen- esis using ampicillm selection. Nuclezc Acids Res....

... Arg522 (NH1)coordinates the chloride ion in tACE]. Arg514 in ACE2 is displaced relative to Arg522 in ACE and issomewhat closer to the zinc-binding site. The CL2binding site is in close proximity ... contains the zinc-bind-ing site, which faces into the deep cleft formed by thetwo subdomains connected at the base of the cleft.The hinge-bending motion observed upon inhibitorbinding occurs ... The zinc protease domain is divided into two sub-domains, which are defined by the movement of thesubdomains relative to each other upon inhibitor bind-ing. Subdomain I (N-terminal) contains...

... purchasedfrom Sigma or Merck. Site- directed mutagenesis Site- directed mutagenesis was performed using a plasmidpT7-7 [13] containing an inserted DNA fragment co dingfor the mature Sfbgly50 (pT7b50) ... (dotted lines) involving the residue 39 and E451 were represented. 4-OH is represented in the equatorial (4e; pointingtowards left) and axial (4a; pointing upwards) positions. 3-OH is always in the ... DDGà, differences in theactivation energy of the g lycosylation steps.Noncovalent interactionsinvolvingamino acid residuesat position 39Noncovalent interactionsinvolving amino acid residuesat...

... the overall substratebinding pocket remains intact. In agreement with data fromthe pH dependence of ligand binding and with the proteincrystal structure, site- directed mutagenesis results emphasizethe ... out a main roleof the side chain of this active site residue in substrate/ligandfixation. The ligand-binding experiments demonstrate thatthe overall substrate-binding pocket remains intact, ... fromPromega Life Sciences. Site- directed mutagenesis reactionswere made using the Altered SitesTMII Kit (Promega LifeSciences).D-amino acids, xanthine, xanthine oxidase, andall other compounds...

... residue Inhibition (%)HgCl2(0.2) Cysteine 100E-64 (0.025) Cysteine 0AEBSF (1.0, 4.0) Serine 0PMSF (1.0) Serine-Cysteine 20TLCK (0.12) Serine-Cysteine 12TPCK (0.2) Serine-Cysteine 0DEPC ... primer walking using the dideoxy chaintermination method [13].For protein purification, the mutated and the wild-typecDNAs were transformed into the E. coli strainM15[pREP4]. An inoculum of ... of PNAE. In order to gain evidence for thesesuggestions, inhibitor studies, site- directed mutagenesis experiments and modelling of PNAE were performed.Inhibitor studiesSeveral known inhibitors...

... nucleotide-binding protein.Nature 250, 194–199.5 Wierenga RK, Terpstra P & Hol WG (1986) Predictionof the occurrence of the ADP-binding bab-fold in pro-teins, using an amino acid sequence ... two enzymes distinguish the cofactors in different ways [3]. In the present study, we have investigated, by meansof site- directed mutagenesis and steady-state kinetics,the individual contribution ... on a Cary 50 recording spectro-photometer (Varian Inc., Palo Alto, CA, USA) by measur-ing the decrease in NAD(P)H concentration via the change in A340 nm, using an extinction coefficient of...

... located in the vicinity of the active site and could form a binding site for a glucosyl unit i n subsite)1 (Fig. 4B). Together with the observations from the site- directed mutagenesis, Trp402 ... solution in the Dali result. It islikely that the inte rface between N-terminal and b-he licaldomains participate in binding of the polysaccharide,pullulan.Comparison of the active sites of ... chains werepredicted t o o rient t o the interface b etween N-terminaland b-helical domains. A structural homology search for theN-terminal domain (residues 25–189) was also carried outusing...

... known to be involved in binding and hydrolysis of the guanidino group ofl-arginine by arginase are strictly conserved in the act-ive site of the agmatinases [19]. Moreover, modelingstudies ... agmatine in E. coli agmatinase and l-arginine in B. caldoveloxarginase [37], indicating that the substrate specificityof these enzymes rely mainly in substituents at C-a.This has been, in fact, ... buffer.Enzyme assays and kinetic studiesRoutinely, enzyme activities were determined by measuringthe formation of urea from l-arginine or agmatine in 50 mm glycine ⁄ NaOH pH 9.0. In studying the effect...