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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME141PlantPlantHormoneHormoneProtocolsProtocolsEditedbyEditedbyGregoryA.TuckerGregoryA.TuckerJeremyA.RobertsJeremyA.RobertsHUMANAPRESSHUMANAPRESSExtractionandPurificationofEnzymes11ExtractionandPurificationofanEnzymePotentiallyInvolvedinABABi
2、osynthesisGregoryA.Tucker,PeteBass,andIanTaylor1.IntroductionTheadventofgeneticengineeringhasprovidedameanstomanipulatethebiosynthesisofplanthormonestotheadvantageofagriculture.Suchmanipulationisverydependentonadetailedknowledgeofthebiosyntheticpathwaysinvolvedintheproduc-tionofthehormones,andmore
3、preciselyabouttheenzymesinvolvedinthisbiosynthesis.Theknowledgeofenzymesassists,butisnotalwaysaprerequisitefor,theisolationofthegeneticmaterial,usuallycDNAs,usedasthegenetictoolstomanipulatehormonesynthesis.Thefirstsuchexampleofthemanipulationofhormonebiosyn-thesisbytheapplicationofgeneticengineer
4、ingwasforthehor-moneethene.ThebiosyntheticpathwayforethenewasfirstdescribedinapplesbyAdamsandYang(1).Methioninewasfoundtobetheprecursorofthishormoneinplanttissueandiscon-vertedviaS-adenysyl-methionine(SAM)intotheuniquecompound1-amino-1-carboxylcyclopropane(ACC).TheconversionofSAMintoACCiscarriedou
5、tbyakeybiosyntheticenzyme,ACCsyn-thase,andthesubsequentconversionofACCintoetheneiscarriedoutbyasecondenzyme,namelyACCoxidase.ThesetwoenzymesFrom:MethodsinMolecularBiology,vol.141:PlantHormoneProtocolsEditedby:G.A.TuckerandJ.A.Roberts©HumanaPressInc.,Totowa,NJ12Tucker,Bass,andTaylorhavebeenextensiv
6、elystudiedandcDNAsforbothidentified,althoughbycompletelydifferentmethods.ACCoxidasecDNAwasinitiallyidentifiedbyaprocessofdifferentialscreeningofatomato-ripeningcDNAlibrary.ThistechniquewasusedtoidentifycDNAclonesfromalibrarymadefromripetomato,whichareexclusivelyexpressedinaripefruitbutnotagreenfru
7、it.Sincethebiosynthesisofethenewasknowntoberipeningspecific,itwaspostulatedthatatleastsomeofanyclonesidentifiedwouldencodebiosyntheticenzymes.Thetechniqueidentifiedseveralclones,oneofwhich,pTOM13,appearedtohaveex
