03-高效蛋白表达系统

高效蛋白质表达系统1.原核表达系统(1)Gramnegative(E.coli)(2)Grampositive(B.subtilis)2.真核表达系统(1)Yeast(2)Baculovirus(3)Drosophila(4)Mammaliancells(COS,CHO,BHKetc.)3.其它(1)Cell-freesystem(2)ProtoblastProteinExpressionProteinexpressionisasubcomponentofgeneexpression.ItconsistsofthestagesafterDNAhasbeentranslatedintoaminoacidchains,whichareultimatelyfoldedintoproteins.Initssimplestform,aproteinexpressionsysteminvolvesatemplate,amechanismoftranscription/translationsuchasacellorcellextract,andtherawmaterialsrequiredtobuildproteins.Proteinexpressioncanbedoneinprokaryotesiftheproteindoesn’tneedtobepost-translationallymodified.Ifperformingstructure-functionstudies,expressinginaeukaryoticsystemisimportantbecausethesecellshavethepropercellularmachinerytodecoratetheproteininquestionwiththecorrectpost-translationalmodifications.Thetypesofeukaryoticcellstypicallyusedinproteinexpressionincludeyeast,insect,andmammalian.ChoiceoftheexpressionsystemCell-freeBacteriaYeastInsectMammalianEasyofuseCostofmediaandEquipmentPos-translationalModifications(Probabilityofproteinfunction)TimeRequirementProkaryoticExpressionProteinexpressioninprokaryoticsystemBacterialexpressionvectorshavesomedistinctfeatures:Induciblepromotersystems;Proteinfusionsincludingfusedtags;Easymanipulation;SomeproblemsofproductioninE.coliInclusionbodies(mostcommoncase)Inclusionbodiesareformedthroughtheaccumulationoffoldingintermediatesratherthanfromthenativeorunfoldedproteins.Itisnotpossibletopredictwhichproteinswillbeproducedasinclusionbodies.Productionofinclusionbodiesarenotdependentontheoriginofproteins,theusedpromoters,thehydrophobicityoftargetproteins...SomeE.coliexpressionhostconsiderationsYeastExpressionYeastExpressionYeastisaeukaryoticorganismthatcanbegrowntoveryhighdensities,whichmakesthemespeciallyusefulfortheproductionofisotopelabeledproteinforNMR.UsefulstrainsincludeSaccharomycescerevisiaeandthemethylotrophicyeastPichiapastoris.Theyeaststrainshavebeengeneticallywellcharacterizedandareknowntoperformmanyposttranslationalmodifications.Yeastcangrowquicklyindefinedmedium,areeasierandlessexpensivetoworkwiththaninsectormammaliancells,andareeasilyadaptedtofermentation.Yeastexpressionisideallysuitedforlarge-scaleproductionofrecombinanteukaryoticproteins.Themajoradvantagesofyeastexpression:highyieldandproductivity,highcelldensities,andsuperiorexpression.ControllableprocessThegrowthmediumthatfeedsyeastiscompletelydefined.Itconsistsofasimple,inexpensiveformulation.Thecarbonsourceisfedtothefermentorataratedesignedtoachievemaximumcelldensitywhilemaintainingoptimalproductionofforeignprotein.Thisprocessminimizesanytoxiceffectstheforeignproteinmighthaveontheyeast.ProductprocessingsimilartomammaliancellsTheyeastexpressionsystemproducesmammalian-likeproteins.Forexample,theexpressionofHepatitisBsurfaceantigen(HBsAg)inyeastleadstoproductionofparticlesthatareimmunoreactivewithanti-HBsAgantibodies.TheseparticlesaresimilartoDaneparticlesisolatedfromtheseraofhumancarriers.StableproductionstrainsExpressionofforeigngenesisachievedbyintegrationofforeignDNAintothechromosomalDNAofhostgenome.TheintegratedDNAisstableformanygenerations;allcellscanproducetheprotein.Incontrast,plasmid-basedsystemsrequireselectivepressureonplasmidstomaintaintheforeignDNA.Cellsthatlosetheplasmidcannotproducethedesiredforeignprotein.DurabilityTheYeastExpressionSystemrequiresnospecialhandling.Itwasdevelopedtowithstandtheadverseconditionsoflargescale,continuousfermentors.ThisfeaturemakesyeastabletosurviveunexpecteddisruptionsinthefermentationprocessLowerproteinproductioncostHighper-cellexpressionlevelscombinedwithhighcell-densitygrowthofyeasttranslatesintogreaterquantitiesofrecombinantproteinperfermentorvolume.Thisreducesproductioncostsbyincreasingtheamountofproductperfermentationrun.Proteinpurificationisanothercost-savingarea.Theyeastsystemcansecreteproteinintothemedium,sothebroththatenterspurificationcontainsahigherconcentrationofthedesiredprotein.Pureproteinisrecoveredwithhigheryieldandlowercost.YeastExpression(Cont.)ExpressioninYeastAutonomousreplicatingvectors-shuttlevectorsExpressioninSaccharomycescerevisiaeAutonomousreplicatingsystemsExpressioninSaccharomycescerevisiaeIntegrativesystemsProbabilityforintegrationhigherwithlinearfragments!ExpressioninSaccharomycescerevisiaeExpressioninS.cerevisiaeVs.PichiapastorisProblemswithproductioninS.cerevisiae:Forsomeproteinsproductionlevelislow.Hyperglycosylation(morethan100mannoseresiduesinN-glycosylation).Sometimessecretionisnotgood-proteinstackincells(periplasma).S.cerevisiaeproduceshighamountofEtOH-toxicforthecells-affectslevelofproduction.AdvantagesofproductioninPichiapastoris:Highlyefficientpromoter,tightlyregulated(alcoholoxidase-AOX,inducedbyMeOH).ProducesnoEtOH-veryhighcelldensity-secretionveryefficient.Secretesveryfewproteins-simplificationofpurificationofsecretedproteins.InsectExpressionBaculovirusorDrosophilaBaculovirusBaculovirusarepresentininvertebratesprimarilyinsectspecies.Theyarenotinfectiousforvertebrates&plants.Genomeiscovalentl