Basic architecture of expression vectors

Introduction to expression vectors. The critical components of expression vectors and their functions

1. ExpressionVectors
TheexpressionvectorisaDNAmoleculethatcarriesaspecificgeneintoahostcelland
usesthecell'sproteinsynthesismachinerytoproducetheproteinencodedbythegene.
Expressionvectorsmaybeplasmidorviral.Plasmidexpressionvectorsareusedfor
highlevelexpressionofproteins.Viralexpressionvectorsareusefulinstudyinggene
underendogenouspromoter,innormalconditions.
Theexpressionvectormustcontainelementsessentialforgeneexpression.Theessential
elementsinclude:strongornormalpromoter,acorrecttranslationinitiationsequence
suchasaribosomalbindingsiteandaninitiationcodon,aterminationcodon,anda
transcriptionterminationsequence.
Additionally,tofacilitateproteinpurificationafterproteinproduction,expressionvectors
usuallyhaveapurificationtag,whichisaddedtotheproteinsequenceofinterest.
Figure:BasicArchitectureofanexpressionvector

2. Componentsofanexpressionvector
a)Selectablemarker:
Intheabsenceofselectivepressureplasmidsarelostfromthehost.
Thesimplestwaytoaddressthisproblemistoexpressfromthesameplasmidan
antibiotic-resistancemarkerandsupplementthemediumwiththeappropriate
antibiotictokillplasmid-freecells.
b)Regulatorygene(repressor):
Manypromotersshowleakinessintheirexpressioni.e.geneproductsareexpressedat
lowlevelbeforetheadditionoftheinducer.
Thiscanbepreventedbytheconstitutiveexpressionofarepressorprotein.
c)Originofreplication:
Theoriginofreplicationcontrolstheplasmidcopynumber.
d)Promoter:
Thepromoterinitiatestranscriptionandispositioned10-100nucleotidesupstreamof
theribosomebindingsite.
Theidealpromoterexhibitsseveraldesirablefeatures:
Itisstrongenoughtoallowproductaccumulationupto50%ofthetotalcellular
protein.
Ithasalowbasalexpressionlevel(i.e.itistightlyregulatedtopreventproduct
toxicity).
Itiseasytoinduce.
e)Transcriptionterminator:
Thetranscriptionterminatorreducesunwantedtranscriptionandincreasesplasmid
andmRNAstability.
f)Shine-Delgarnosequence:
TheShine-Dalgarno(SD)sequenceisrequiredfortranslationinitiationandis
complementarytothe3'-endofthe16SribosomalRNA.
Theefficiencyoftranslationinitiationatthestartcodondependsontheactual
sequence.Theconcensussequenceis:5'-TAAGGAGG-3'.
Itispositioned4-14nucleotidesupstreamthestartcodonwiththeoptimalspacing
being8nucleotides.

3. Toavoidformationofsecondarystructures(whichreducesexpressionlevels)this
regionshouldberichinAresidues.
g)Startcodon:
Startcodonisinitiationpointoftranslation.InE.colithemostusedstartcodon
isATG.GTGisusedin8%ofthecases.TTGandTAAarehardlyused.
h)Tagsandfusionproteins:
N-orC-terminalfusionsofheterologousproteinstoshortpeptides(tags)ortoother
proteins(fusionpartners)offerseveralpotentialadvantages:
Improvedexpression.FusionoftheN-terminusofaheterologousproteintotheC-
terminusofahighly-expressedfusionpartneroftenresultsinhighlevelexpressionof
thefusionprotein.
Improvedsolubility.FusionoftheN-terminusofaheterologousproteintotheC-
terminusofasolublefusionpartneroftenimprovesthesolubilityofthefusionprotein.
Improveddetection.Fusionofaproteintoeitherterminusofashortpeptide(epitope
tag)orproteinwhichisrecognizedbyanantibodyorabindingprotein(Westernblot
analysis)orbybiophysicalmethods(e.g.GFPbyfluorescence)allowsfordetection
ofaproteinduringexpressionandpurification.
Improvedpurification.Simplepurificationschemeshavebeendevelopedfor
proteinsfusedateitherendtotagsorproteinswhichbindspecificallytoaffinity
resins.
i)Proteasecleavagesite:Proteasecleavagesitesareoftenaddedtobeabletoremoveatag
orfusionpartnerfromthefusionproteinafterexpression.However,cleavageisrarely
completeandoftenadditionalpurificationstepsarerequired.
j)Multiplecloningsite:Aseriesofuniquerestrictionsitesthatenablestoclonegeneof
interestintothevector.
k)Stopcodon:Terminationoftranslation.Thereare3possiblestopcodonsbutTAAis
preferredbecauseitislesspronetoread-throughthanTAGandTGA.Theefficiencyof
terminationisincreasedbyusing2or3stopcodonsinseries.

4. Table:Componentsofexpressionvector
Goal Component
1.Insertcargointotheplasmidandverifythe
insertsequenceaccuracy
•MCS–restrictionsitesORrecombination
regions
•5’and3’Primersitesforsequence
verification
2.Insertplasmidintocells,enabletheplasmid
toreplicateinsidethehost&selectforcells
carryingtheplasmid
•Backbonecompatiblewithcloningmethod
•Originofreplication
•Selectionmarkerand/orscreeningmarker
3.TranscribemRNAfromtheplasmid
•Promoter(constitutiveorinducible)
operator,terminator
4.TranslatemRNAintoprotein
•RibosomeBindingSite,startcodon,stop
codon
5.Promoteproperfoldingofnascentprotein
•co-expressionofchaperones
•Solubilizationtags
•custom-designedsyntheticRBS
•Codon-optimizedORF
6.DetectorPurifytargetprotein
•Epitopetags(His)
•reporters(GFP)