EXPRESSION SYSTEMS.ppt

Expression Systems - for protein production

1. HOSTS FOR GENE CLONING
Dr. Esther Shoba R
Assistant Professor
Department of Life Sciences
Kristu Jayanti College

2. INTRODUCTION
•Thedevelopmentofgeneticengineeringandcloninghasopened
manypossibilitiesofexpressionandisolationofheterologous
proteinsforresearchpurposes.
•Largescaleapplicationssuchasenzyme,antibodyorvaccine
production,theamountofproteinrequiredisconsiderablyhigh.
•Insuchcasesthesysteminwhichtheproteinisexpressedmustbe
easytocultureandmaintain,growrapidly,andproducelarge
amountsofprotein.
•Theserequirementsledtothediscoveryofproteinexpression
systems.Thevariousproteinexpressionsystemsarebacteria,yeast,
insectormammaliansystems.

3. •Thefollowingfactorsdeterminethetypeof
expressionsystemusedtoproducerecombinant
proteins:
•timespentinexpressingtheprotein
•easeofhandlingtheexpressionsystem
•amountofproteinneeded
•massoftheprotein
•typeofpost-translationalmodifications,numberof
disulfidebonds
•destinationoftheexpressedprotein

4. •Theprocessofexpressingarecombinantprotein
inanexpressionsystemrequiresthefollowing
information/components.
•identificationofthegenethatencodesthe
proteinofinterest
•generationofcDNAfromtherespectivemRNA
•selectionofsuitableexpressionvectortoinsert
thegenesequence
•selectionofsuitablesystemthatcanexpressthe
vector
•appropriatescreeningandscalingupmethods

6. Bacterial protein expression systems –
Escherichia coli
•Bacteriaactasrapidandsimplesystemsofexpressingrecombinant
proteinsduetotheshortdoublingtime.
•Themediarequiredtoculturethemarenotexpensiveandthe
methodsadaptedtoscale-upbioproductionarestraightforward.
•
•ThemostwidelyusedhostsystemisE.colisincethereisample
knowledgeaboutitsgenetics,genomesequenceandphysiology.
•Thegeneticmanipulationiseasyanditalsogrowstohighdensities
andissuitableforlarge-scalefermentations.
•However,thecellwallofE.colicontainstoxicpyrogensandthe
expressedproteinsmayhavetobeextensivelytestedbeforeuse.

8. •Features
•low cost culture methods
•flexible system –can carry plasmids with
multiple promoters, tags and restriction sites
•easy to scale up and produce higher yield of
protein

9. DH5 ALPHA
•DH5alphaisthemostfrequentlyusedE.colistrain
forroutinecloningapplications.Inadditionto
supportingblue/whitescreeningrecA1andendA1
mutationsinDH5aincreaseinsertstabilityand
improvethequalityofplasmidDNApreparedfrom
minipreps.
•Application:
•Highesttransformationefficiency
•Generalcloning
•Blue-whiteselection
•Plasmidisolation

10. •TherecA1mutationisasinglepointmutationthat
replacesglycine160oftherecApolypeptidewith
anasparticacidresidueinordertodisablethe
activityoftherecombinasesandinactivate
homologousrecombination.
•TheendA1mutationinactivatesanintracellular
endonucleasetopreventitfromdegradingthe
insertedplasmid.
•High insert stability due torecA1 mutation
•High yield and quality of DNA due toendA
mutation

11. BL 21 HOST CELLS
•BL21 CompetentE. coliis awidely used non-
T7 expressionE. colistrain and is suitable for
transformation and protein expression.
•Ideal for P
lac, P
tacexpression vectors
•Protease deficient

12. BL21 DE 3pLysS
•BL21(DE3)pLysSisaderivativeofBL21thathastheT7
RNApolymerasegeneunderthecontrolofthelacUV5
promoter.
•Thisarrangementisonaphagegenome,calledDE3.
•DE3isinsertedintothechromosomeofBL21tomake
BL21(DE3).
•pLysSisaplasmidthatcontainstheT7lysozymegene
(LysS).
•TheT7lysozymebindstoT7RNApolymerasecausing
inhibitionuntilinductionbytheadditionofIPTG.
•WhenIPTGisadded,theamountofT7RNA
polymeraseincreasesandovercomestheinhibitionby
LysS.

14. •Proteinexpressionfromhigh-copynumberplasmidsand
powerfulpromoterswillgreatlyexceedthatofanynative
hostprotein,usingupvaluableresourcesinthecellthus
leadingtoslowedgrowth.
•Additionally,someproteinproductsmaybetoxictothehost
whenexpressed,particularlythosethatareinsoluble,acton
DNA,orareenzymaticallyactive.
•Forthisreason,recombinantproteinsaretypicallyexpressed
inE.coliengineeredtoaccomodatehighproteinloadsusing
induciblepromotersystems

16. •ompT:Strainsharboringthismutationaredeficientinouter
membraneproteaseVII,whichreducesproteolysisoftheexpressed
recombinantproteins.
•lonprotease:Strainswherethisiscompletelydeleted(designated
lonorΔlon)similaryreduceproteolysisoftheexpressedproteins.
•hsdSB(rB-mB-):Thesestrainshaveaninactivatednative
restriction/methylationsystem.Thismeansthestraincanneither
restrictnormethylateDNA.
•dcm:Similarly,strainswiththismutationareunabletomethylate
cytosinewithinaparticularsequence.

17. Yeast protein expression systems –Saccharomyces cerevisiae
•Thehighlydevelopedgeneticsystem,easeofuse,reduced
timeinputandcostshavemadeS.cerevisiaeanattractive
organismfortheexpressionandproductionofrecombinant
proteins.
•Yeastsareabletocarryspecificallydesignedplasmidsandthis
abilityisvaluableinarecombinantproteinexpressionsystem.
•Theplasmidusedconsistsofrestrictionsitesthatcanbeused
toinsertthegenesequenceofinterest.
•Transformationofyeastswiththeplasmidproducesthe
desiredproteinandcanbeappropriatelyscaledup.

18. •ExpressionofproteinusingS.cerevisiaeinvolvesthefollowing
steps:
•useofcompetentE.colicellstotakeupDNAsequenceofinterest
•integrationoftheDNAintobacterialgenomeorcircularizationof
theDNAsequencetoexistasaplasmid
•selectionoftransformedE.coliusingaselectionmarker
(antibiotic)
•expansionofselectedE.coliinappropriateculturemedia,suchas
classicLBoptions
•isolationofDNAorplasmid
•transformationintoyeast
•screenthetransformantsforintegrationofDNAintoyeast
chromosome
•selectionandscaling-upofhighexpressingyeastclonesin
appropriateculturemedia
•isolationandpurificationofintracellular/secretedproteins

19. •Features
•lowcostculturemethods
•suitableforbothintracellularandsecreted
proteins
•provideseukaryoticpost-translational
glycosylationofproteinsalthoughitresultsin
high

20. •AyeastcommonlyusedforproteinproductionisPichia
pastoris.ExamplesofyeastexpressionvectorinPichiaarethe
pPICseriesofvectors,andthesevectorsusetheAOX1
promoterwhichisinduciblewithmethanol.
•Theplasmidsmaycontainelementsforinsertionofforeign
DNAintotheyeastgenomeandsignalsequenceforthe
secretionofexpressedprotein.Proteinswithdisulphide
bondsandglycosylationcanbeefficientlyproducedinyeast.
•AnotheryeastusedforproteinproductionisKluyveromyces
lactisandthegeneisexpressed,drivenbyavariantofthe
stronglactaseLAC4promoter