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Oxford Genetics/pSF-T7-NH2-OmpA-SP3 (OG164) Bacterial Secretion Plasmid/OG164/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-T7-NH2-OmpA-SP3
ProductCode:OG164
Size(bp):3938bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:T7PolymerasePromoter
PlasmidPurpose:
Thisvectorallowstranscriptionofun-polyadenylatedandun-cappedRNAusingtheT7bacteriophagepolymerase.ThisvectoralsocontainstheEscherichiacoli(E.coli)outermembraneproteinA(OmpA)secretorysignalpeptide.WhenpositionedattheN-terminusofaproteinitshoulddirecttheproteintobesecretedoutofthebacterialcytosolandintotheperiplasmicspace.TheOmpAsecretorytagis21aminoacidsinlength.ThesignalpeptideproteinsequenceisMKKTAIAIAVALAGFATVAQA.CleavageoccursafterthefinalAlanineresidue.Thepeptidetagwillbecleavedthematureproteinduringexport.
Thistechniquecanbeusedtoincreasedi-sulphidebondformationandalsoreducesproteolyticdegradation.ThismethodalsoreducescontaminationfromendogenousproteinsbecauseveryfewE.coliproteinsaresecreted.Asubfractionoftheproteinsecretedintotheperiplasmicspacecanalsoextractedfromthegrowthmedia.ItisstillunclearwhythisoccursbutitbelievedtoreflectincreasedpermeABIlityoftheoutermembraneparticularlyduringextendedcultureperiods.
SecretionofproteinsinE.colicansometimesbedifficult.Potentialissuesincludeproteintoxicitywithintheperiplasmvariablesecretionefficiencyformationofinclusionbodieswithinthecytoplasmwhenusingstrongpromotersincorrectdis-sulphideformation.BackgroundtobacterialsecretionsystemsincludingpossIBLesolutionstotheseproblemscanbefoundinChoietal2004AppliedMicroBIOLOGyandBiotechnology64(625-635).
AllSnapFastTMvectors(exceptthoseintheterminatorscategoryonourwebsite)willcontainaT7terminatordownstreamofthemultiplecloningsite.Thiswillenabletranscriptiontermination.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
CloninginaGene:Thisvectorhasbeendesignedtoallowtheadditionofapeptidetagtotheendofaproteinofinterestusingstandardcloningtechniques.
MultipleCloningSiteNotes:ThereisastartcodonintheNcoIsitecanberemovedbydigestionwithKpnIifrequired.TheMCSforgeneinsertionsextendsfromNotItoXbaIhoweverthetagresidesbetweentheNotIandHindIIIsites.ThereareShine-DalgarnosequencesandKOZAKsequencesalignedwiththestartcodonofthepeptidetag.
TheClaItoNheIsiteshaveotherfunctionssuchasaddingC-terminalpeptidetagssecondpromotersorIRESexpressioncomponents.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofC-terminalpeptidetagssequencesifthestopcodonisplacedinthisposition.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
