ovguide

InstructionManual

GuidetoBaculovirus

ExpressionVectorSystems

(BEVS)andInctCellCulture

Techniques

INSIDEFRONTCOVER

i

uction

OverviewofBaculovirology............................................................................................................................................1

BaculovirusasExpressionVectors............................................................................................................................1

AdvantagesofBEVSTechnology...................................................................................................................................2

GeneratingaRecombinantVirusbyHomologousRecombination.................................................................................3

GeneratingaRecombinantVirusbySite-SpecificTransposition....................................................................................3

InctCellCultureTechniques.......................................................................................................................................4

olsforCulturingHostCells.............................................................................................................................6

Protocol1:SubculturingMonolayerCultures............................................................................................................6

Protocol2:AdaptingMonolayerCellstoSuspensionCulture..................................................................................6

Protocol3:MaintainingSuspensionCultures...........................................................................................................7

Protocol4:AdaptingCulturestoSerum-FreeMedium.............................................................................................8

Protocol5:PreparingaMasterCellSeedStock......................................................................................................8

olsforGeneratingaRecombinantBaculovirus..........................................................................................10

Protocol6:IsolationofBacmidDNAforBAC-TO-BAC®BaculovirusExpressionSystem

withtheCONCERT™HighPurityPlasmidPurificationSystem................................................................10

Protocol7:CationicLiposome-MediatedTransfectionUsingCELLFECTINReagent.............................................11

Protocol8:VirusPlaqueAssay...............................................................................................................................11

olsforPurifyingandProducingRecombinantAcNPVandProtein.........................................................14

Protocol9:PlaquePurificationofRecombinantViralClones.................................................................................14

Protocol10:AmplifyingtheVirusStock....................................................................................................................14

Protocol11:IdentifyingPlaquesbyNeutralRedStaining........................................................................................14

Protocol12:OptimizingVirusStockProduction.......................................................................................................15

Protocol13:HarvestingtheVirus.............................................................................................................................15

Protocol14:ConcentratingtheVirus........................................................................................................................15

Protocol15:StoringtheVirus...................................................................................................................................16

Protocol16:OptimizingHeterologousProteinProduction........................................................................................16

ingRecombinantProteins...............................................................................................................................17

PurifyingSecretedProteins...........................................................................................................................................17

PurifyingIntracellularProteins.......................................................................................................................................18

nces................................................................................................................................................................21

dProducts........................................................................................................................................................22

AppendixA:ApplicationsDataforInctCellLinesGrowninSerum-FreeMedia.......................................................23

Figures:

1Invivobaculovirusinfectionandreplication........................................................................................................................2

2Generatingarecombinantbaculovirusbyhomologousrecombination...............................................................................3

3GenerationofrecombinantbaculovirusandgeneexpressionwiththeBAC-TO-BACexpressionsystem........................4

TableofContents

ii

Tables:

1InctcelllinescommonlyudinBEVSapplications.......................................................................................................4

2InctcellculturemediacommonlyudinBEVSapplications.........................................................................................4

3Effectsofrumoncellcultures..........................................................................................................................................5

4Ufulmediumvolumes......................................................................................................................................................7

5Troubleshootingvirusplaqueassays................................................................................................................................13

6RecommendedmaximuminfectiondensitiesfortheproductionofrAcNPVorrecombinantproducts.............................14

7Maximumcelldensitiesinsmall-scalesuspensionculture...............................................................................................22

8β-galactosidaexpressioninsmall-scalesuspensionculture.........................................................................................23

9Pilot-scalerecombinantproteinexpressionincellscultured.............................................................................................23

10rAcNPVtitersinsmall-scalesuspensionculture...............................................................................................................23

BAC-TO-BAC®,CELLFECTIN®,DH10BAC™,pFASTBAC™1,EXPRESS-FIVE®,CONCERT™,TECH-LINESM,andtheLifeTechnologieslogoaremarksofLifeTechnologies,Inc.

TheBAC-TO-BACBaculovirusExpressionSystemissoldunderpatentlicenforrearchpurposonly,andnolicenforcommercialuisincluded.

RequestsforcommercialmanufactureorushouldbedirectedtotheOfficeroftheDirector,MailZone02A,MonsantoCorporateRearch,rgh,

,MO63167.

High-Five™isatrademarkofInvitrogenCorporation.

PLURONIC®isaregisteredtrademarkofBASFCorporation.

Falcon®isaregisteredtrademarkofBectonDickinson&Company.

levirionsare

producedandsurroundedbyacrystallinepolyhedra

usparticlesproducedinthenucleusare

embeddedwithinthepolyhedringeneproductanda

carbohydrate-richcalyx.

Infection

Figure1summarizeshowbaculovirusinfectcellsand

the

lyticcycle,envelopedandbuddedvirionsaregenerated.

Thevirionspromotehorizontaltransmissionoftheinfec-

tionthroughoutthetissueinaninvivoinfectionofaworm

larvae,orthroughoutthecellcultureinaninvitroover-

o,thiscycleixploitedtoboth

generatevirusstocksandestablishafullydevelopedinfec-

the

occludedcycle,virionspackagedinthePIBsaregenerated.

Invivo,thevirionspromoteverticaltransmissionofthe

o,apolyhedrin

genemodifiedtoexpressarecombinantgeneproductin

mically,theesntialdiffer-

encebetweenthelyticandoccludedcyclesistheinduction

ofpolyhedrinproductionatthebeginningoftheverylate

pha.

Youneedtobeabletodistinguishbetweentheinitiation

ofvirusproductionandbudding,atapproximately8to10h

post-infection,andtheinitiationofproteinexpressionunder

controlofthepolyhedrinpromoter,atapproximately20to24

gso,youwillbeabletoeffcientlyproducehigh-

titerbaculovirusstocksandhigh-qualityrecombinant

product(i.e.,productthatisnon-degradedandfreeofcell

debris).

VerticalTransmission

AftertheOVisingestedbyinctlarvae,thecrystalline

polyhedrinmatrixisdegradedinthealkalinemid-gutofthe

edvirionsarereleadandfutomicro-

edcellsreleaEVfromthe

bamentmembranesideofthemid-gutcellintothe

hemolymphsystem.

HorizontalTransmission

EVenterstheincthemocoelandimmediatelyspreads

throughouttheinct’sopencirculatorysystem,infecting

10viralgenerations,theinctdies

andtheOV,producedduringtheverylatestageofinfection,

isreleadintotheenvironment.

BaculovirusasExpressionVectors

Themajordifferencebetweenthenaturallyoccurringin

vivoinfectionandtherecombinantinvitroinfectionisthat

thenaturallyoccurringpolyhedringenewithinthewild-type

baculovirusgenomeisreplacedwitharecombinantgeneor

enesarecommonlyunderthecontrolof

atephaofinfection,

thevirionsareasmbledandbuddedrecombinantvirions

r,duringtheverylatephaofinfec-

tion,theinrtedheterologousgenesareplacedunderthe

transcriptionalcontrolofthestrongAcNPVpolyhedrin

,recombinantproductixpresdinplace

1

Recombinantbaculovirusarewidelyudto

expressheterologousgenesinculturedinctcells

ge-scaleapplications,the

baculoviruxpressionvectorsystem(BEVS)isparticularly

lizedmedia,transfectionreagents,

andvectorshavebeendevelopedinrespontorecent

advancesininctcellcultureandmolecularbiologymeth-

ods.

Thefollowingareimportantchoicesindesigninga

systemforrecombinantproteinproduction:

•Selectingtheexpressionvector,includingthestyleor

typeofpromoter,thatprovidesbestresultswiththe

recombinantgeneproductbeingexpresd.

•Evaluatinginctcelllines,growthmedia(rum-

supplementedorrum-free),andfeeding/infection

strategiesthatallowforoptimalrAcNPVand/orproduct

expression.

•Choosingascalableprocessofcellcultureanddeciding

onotherfactorsaffectingdownstreamprocessing.

OverviewofBaculovirology

Baculovirusarethemostprominentvirusknownto

edouble-stranded,

circular,supercoiledDNAmoleculesinarod-shapedcapsid

(1).Morethan500baculovirusisolates(badonhostsof

origin)havebeenidentified,mostofwhichoriginatedin

arthropods,particularlyinctsoftheorderLepidoptera

(2,3).Twoofthemostcommonisolatesudinforeign

geneexpressionareAutographacalifornicamultiplenuclear

polyhedrosisvirus(AcMNPV)andBombyxmori(silkworm)

nuclearpolyhedrosisvirus(BmNPV).

Wild-typebaculoviruxhibitbothlyticandoccluded

lifecyclesthatdevelopindependentlythroughoutthethree

lowingarecharacteristics

ofthethreephas:

ha:Inthispha(alsoknownasthevirus

synthesispha),theviruspreparestheinfectedcellfor

ncludeattachment,pene-

tration,uncoating,earlyviralgeneexpression,andshut

initialviralsynthesis

occurs0.5to6hafterinfection.

a:Inthispha(alsoknownastheviralstruc-

turalpha),lategenesthatcodeforreplicationofviral

n6

and12hafterinfection,thecellstartstoproduceextra-

cellularvirus(EV),alsocallednon-occludedvirus(NOV)

orbuddedvirus(BV).TheEVcontainstheplasma

membraneenvelopeandglycoprotein(gp)64necessary

leaofextracel-

lularvirusoccurs18to36hafterinfection.

tePha:Inthispha(alsoknownastheviral

occlusionproteinpha),thepolyhedrinandp10genes

areexpresd,occludedvirus(OV)—alsocalledocclu-

sionbodies(OB)orpolyhedralinclusionbodies

(PIBs)—areformed,n24

and96hafterinfection,thecellstartstoproduceOV,

whichcontainsnuclearmembraneenvelopesandthe

uction

bly,production,andexpressionofrecombinantgene

layercultures,areasofinfectiondisplay

,in

suspensioncultures,celldensitiesbegintodecrea.

Infectedcellscontinuetobeincreadindiameterand

oplasmmaycontain

vacuoles,andthenucleimaydemonstrategranularity.

Astheinfectedcellsdie,plaquesdevelopinimmobilized

quescanbeidentifiedunderamicro-

scopeasregionsofdecreadcelldensity,orbyeyeas

regionsofdifferentialrefractivity.

AdvantagesofBEVSTechnology

Since1983,whenBEVStechnologywasintroduced,the

baculovirussystemhasbecomeoneofthemostversatile

andpowerfuleukaryoticvectorsystemsforrecombinant

proteinexpression(4).Morethan600recombinantgenes

1985,

whenthefirstprotein(IL-2)wasproducedinlargescale

fromarecombinantbaculovirus,uofBEVShas

increaddramatically(5).Baculovirusofferthefollowing

advantagesoverotherexpressionvectorsystems.

•Safety:Baculovirusareesntiallynonpathogenicto

mammalsandplants(6).Theyhavearestrictedhost

range,whichoftenislimitedtospecificinvertebrate

etheinctcelllinesarenottrans-

formedbypathogenicorinfectiousvirus,theycanbe

celllinesorhelpervirusarenotrequiredbecauthe

baculovirusgenomecontainsallthegeneticinformation.

•EaofScaleUp:Baculovirushavebeenrepro-

duciblyscaledupforthelarge-scaleproductionof

biologicallyactiverecombinantproducts.

2

y,the

recombinantproteinsareprocesd,modified,andtargeted

totheappropriatecellularlocations.

Cytopathogenesis

Astherecombinantinfectionadvances,veralmorpho-

ingofthe

infectioncycleandthechangesincellmorphologyvarywith

a-

bolicconditionofthecultureandgrowthmediumudalso

lowing

morphologicalchangesaretypicalofmonolayerSf9cells

infectedwithrecombinantAcNPV.

ha:Infectionbeginswiththeadsorptive

endocytosisofoneormorecompetentvirionsbyacell

inahighmetabolicstate(peakreplicationrate).The

nucleocapsidspassthroughthecytoplasmtothe

evirionnterthenucleus,they

30minof

infection,thefirst6hof

infection,thecellularstructurechanges,normalcellular

functionsdeclineprecipitously,andearly-phaproteins

becomeevident.

a:Within6to24hafterinfection,aninfected

cellceasmanynormalfunctions,stopsdividing,and

islogarithmicallyincreasingproductionofviralgenome

ogenicstroma(anelectron-

dennuclearstructure)becomeswelldeveloped.

Infectedcellsincreaindiameterandhaveenlarged

lsmaydemonstratereducedrefractivity

edculturesstop

growing.

tePha:Within20to36hafterinfection,cells

ceaproductionofbuddedvirusandbegintheasm-

baculovirusinfectionandreplication.

Inctskilledbyabaculo-

virusinfectionreleapoly-

hedraontoplantsurfaces.

Thepolyhedraontheplantsurfaceareingestedby

edpolyhedraaretransportedtothe

mid-gutwheretheyaredissolvedandthevirus

sfuwithmid-gutcellmembranesand

nucleocapsidsaretransportedtothenucleusandviral

replicationcommences.

~8hlater,maturebuddedvirus

particlesarereleadintothe

hemolymph,wheretheymayinfect

virusparticles

areoccludedintopolyhedra.

~7to14dayslater,cellslyand

theinctdies.

•HighLevelsofRecombinantGeneExpression:In

manycas,therecombinantproteinsaresolubleand

easilyrecoveredfrominfectedcellslateininfection

whenhostproteinsynthesisisdiminished.

•Accuracy:Baculoviruscanbepropagatedininct

hostswhichpost-translationallymodifypeptidesina

mannersimilartothatofmammaliancells.

•UofCellLinesIdealforSuspensionCulture:

AcNPVisusuallypropagatedincelllinesderivedfromthe

fallarmywormSpodopterafrugiperdaorfromthe

nesareavailable

thatgrowwellinsuspensioncultures,allowingthe

productionofrecombinantproteinsinlarge-scalebioreac-

tors.

GeneratingaRecombinantVirusby

HomologousRecombination

Usinghomologousrecombinationtogeneratearecom-

tcommon

hasalarge(130-kb),circular,double-strandedDNA

eofinterestisclonedintoatransfervector

containingabaculoviruspromoterflankedbybaculovirus

DNAderivedfromanonesntiallocus—inthisca,the

eofinterestisinrtedintothe

genomeoftheparentvirus(suchasAcMNPV)byhomolo-

gousrecombinationaftertransfectionintoinctcells.

Typically,0.1%to1%oftheresultingprogenyarerecombi-

ombinantsareidentifiedbyalteredplaque

ctorwiththepolyhedrinpromoter,asin

thixample,thecellsinwhichthenucleidonotcontain

occludedvirus,ionofthe

desiredocclusion-minusplaquephenotypeagainstthe

backgroundofgreaterthan99%wild-typeparentalvirus

isdifficult.

Ahigherpercentageofrecombinantprogenyvirus

(nearly30%higher)resultswhentheparentvirusis

linearizedatoneormoreuniquesiteslocatednearthe

targetsiteforinrtionoftheforeigngeneintothe

baculovirusgenome(7,8).Toobtainanevenhigherpropor-

tionofrecombinants(80%ormore),linearizedviralDNA

thatismissinganesntialportionofthebaculovirus

genomedownstreamfromthepolyhedringenecanbeud

(9).Theapproachescantakemorethanamonthtopurify

plaques,amplifythevirus,andconfirmthedesiredrecom-

binants.

GeneratingaRecombinantVirusbySite-

SpecificTransposition

Afasterapproachforgeneratingarecombinant

baculovirus(10,11)ussite-specifictranspositionwithTn7

toinrtforeigngenesintobacmidDNApropagatedinE.

eofinterestisclonedintoapFASTBAC™vector,

andtherecombinantplasmidistransformedintoDH10BAC™

competentcellswhichcontainthebacmidwithamini-attTn7

i-Tn7elementon

thepFASTBACplasmidcantranspotothemini-attTn7

targetsiteonthebacmidintheprenceoftransposition

escontain-

ingrecombinantbacmidsareidentifiedbyantibiotic

lectionandblue/whitescreening,sincethetransposition

resultsindisruptionofthelacZαlecular

clonescontainingtherecombinantbacmid,andthisDNAis

pstogeneratea

recombinantbaculovirusbysite-specifictranspositionusing

theBAC-TO-BAC™BaculovirusExpressionSystemare

outlinedinfigure3.

AvarietyofpFASTBACdonorplasmidsareavailable

smidpFASTBAC1

(11)isudtogenerateviruswhichwillexpressunfud

STBACHTriesofvectors

(12)areudtoexpresspolyhistidine-taggedproteins

pFASTBACDUALvectorhastwopromotersandcloning

sites,allowingexpressionoftwogenes,onefromthepoly-

hedrinpromoterandonefromthep10promoter.

AdvantagesofSite-SpecificTransposition:Usingsite-

specifictranspositionhastwomajoradvantagesover

homologousrecombination:

3

tingarecombinantbaculovirusby

homologousrecombination.

Clonethegenetobeexpresd

intothetransfervector

DAY0:

Co-transfectinctcellswithwild-typeAcMNPV

DNAandrecombinanttransfervector(Protocol7)

DAY26:

Amplifythevirus(2to3rounds)

(Protocol10)

DAY5:

Fromalow-titer(1×102to1×104)recombinant

viralstock,purifythedesiredrecombinantwith3

roundsofplaqueassays(Protocols8and9)

DAYS43–50:

Expresstheprotein

DAYS40–47:

Fromahigh-titer(1×107to1×108

pfu/ml)recombinantbaculovirusstock,

infecttheinctcells(Protocol16)

•One-StepPurificationandAmplification:Becau

recombinantvirusDNAisolatedfromlectedcolonies

isnotmixedwithparental,nonrecombinantvirus,multi-

pleroundsofplaquepurificationarenotrequiredand

7to10

days,youwillhavepurerecombinantvirustitersof

>1×107pfu/mlwithoutanyviralamplification.

•RapidandSimultaneousIsolationofMultiple

RecombinantVirus:Thisfeatureisparticularlyvalu-

ableforexpressingproteinvariantsinstructure/function

studies.

InctCellCultureTechniques

Successfulcultureofinctcellsrequiresabasicfamil-

iaritywithinctcellphysiologyandgeneralcellculture

erialsandmethodsforuwithinctcell

culturehaveevolvedandcontributedtotheadvancementof

lowingfactorshavebeensignifi-

cant:

•Growthsupplementsandshearforceprotectantsare

widelyud.

•Serum-freemedia(SFM)havereplacedrum-supple-

mentedmedia,particularlyforlarge-scaleproduction.

•Someinctcelllineshavebeenoptimizedforuin

suspensionculture,especiallyufulforscale-up.

CellLines

ThemostcommoncelllinesudforBEVSapplications

e,Sf9,aclonalisolateofthe

4

tionofrecombinantbaculovirusandgeneexpressionwiththeBAC-TO-BACexpressionsystem.

DetermineViralTiter

byPlaqueAssay

Recombinant

Baculovirus

Particles

Transfectionof

InctCellswith

CELLFECTINReagent

Transformation

Donor

Foreign

Gene

p

P

o

l

h

Tn7R

Tn7L

Recombinant

DonorPlasmid

pFASTBACdonorplasmid

CloneGeneofInterest

Transposition

AntibioticSelection

o

r

Infectionof

InctCells

Mini-prepofHigh

molecularWeightDNA

Recombinant

BacmidDNA

RecombinantGeneExpression

orViralAmplification

DAYS2–3DAY1

DAY4DAYS5–7

Donor

Helper

B

a

c

m

i

d

mi

ni

-

a

t

t

T

n

7

l

a

c

Z

lls

pPolh

(Lac7

-

)

ContainingRecombinantBacmid

Foreign

Gene

Helper

SpodopterafrugiperdacelllineIPLB-Sf21-AE,isprobably

9wasoriginallyestablishedfrom

ovariantissueofthefallarmyworm(13).Althoughthereis

significantscientificdataonthecharacteristicsofthis

Lepidopterancellline,itremainstobeconfirmedwhetherit

isthebestlineforvirusorrecombinant

grearchsuggeststhatdiffer-

entinctcelllinesmaysupportvaryinglevelsof

expressionanddifferentialglycosylationwiththesame

recombinantprotein(14).

celllinescommonlyud

inBEVSapplications.

InctSpeciesCellLine

SpodopterafrugiperdaSf9

SpodopterafrugiperdaSf-21

TrichoplusianiTn-368

TrichoplusianiHigh-Five™BTI-TN-5B1-4

Note:Eachofthecelllineshasbeensuccessfullyadaptedtosuspensioncultures.

MediaandGrowthSupplements

Commonlyudinctcellculturemediaarelistedin

ionally,Grace’sSupplemented(TNM-FH)

mediumhasbeenthemediumofchoiceforinctcell

r,otherrum/hemolymph-dependentand

rum-freeformulationshaveevolvedsinceGrace’s

mediumwasintroduced.

cellculturemediacommonlyud

inBEVSapplications.

Serum/hemolymph-dependentmediaSerum-freemedia

Grace’sSupplemented(TNM-FH)Sf-900IISFM

IPL-41EXPRESS-FIVE™SFM

TC-100

Schneider’sDrosophila

Note:Storeliquidmediawhichallcontainphotolabilecomponentsinthedarkat4°Cto8°C.

Fetalbovinerum(FBS)hasbeentheprimarygrowth

almostcompletelysupplantedthefirstmajorsupplement,

incthemolymph,whichtendedtomelanizeanddeterio-

ratethequalityoftheculturemedium(15).Ofthemorethan

100inctcellculturemediadescribedintheliterature,a

majoritycontain,orrecommend,varyingconcentrationsof

rumasagrowthsupplement(16).

Supplementationwithrumhasbothdesirableand

resummarizedintable3.

Serumandotherundefinedsupplements,suchaslactalbu-

minhydrolysateandyeastolate,providecellswith

growth-promotingfactorssuchasaminoacids,peptides,

andvitamins,whichmaynotbeavailableindefined,basal

mediaformulations.

sofrumoncellcultures.

DesirableEffectsUndesirableEffects

PromotesgrowthMaycauexcessivefoaminginsparged

bioreactors

ProvidesshearforceprotectionMayintroduceadventitiousagents

Protectsagainstproteolyticdegrad-Increascostandcomplexityofdownstream

ationandenvironmentaltoxicitiesprocessing

Fluctuatesinprice,quality,andavailability

ContributescellularattachmentMaydemonstratesuboptimalcellgrowthor

factorstoxicity

Maydemonstratedecreadproductyields

Before1984,fewscientificarticlesreferencedrum-

time,rum-freeinct

culturemediawereudmostlytoreplicateinctvirus

arlySFMformula-

tionswerenotwellsuitedforuinproducingrecombinant

ormulationscontainedinherentflawsthat

limitedcellulargrowth,suspensionculture,andprotein

Sapplications,theearlyformulations

weregenerallypoorlydefinedandtoorichinprotein.

Mostcommerciallyavailablerum-freeinctmedia

areesntiallysimplevariationsofIPL-41basalmedium

supplementedwithundefinedproteinhydrolysatesanda

lipid/surfactantemulsion(17).Second-generationrum-

freeformulationssuchasSf-900IISFMandEXPRESS-FIVE

SFMarespecificallydesignedforlarge-scaleproductionof

ntainoptimizedconcentra-

tionsofaminoacids,carbohydrates,vitamins,andlipidsthat

reduceoreliminatetheeffectofrate-limitingnutritional

-900IISFMand

EXPRESS-FIVESFMsupportfasterpopulationdoublingtimes

andhighersaturationcelldensitiesthandotraditional

,youcanobtainbothhigherwild-typeorrecom-

binantbaculovirustitersandincreadlevelsoryieldsof

recombinantproteinexpressionbyusingtheformula-

imizedformulationsofferthefollowingadvan-

tagesoverra:

•Eliminatetheneedforcostlyfetalbovineandother

animalrasupplements

•Increacellandproductyields

•Eliminateadventitiousagents

•Havelot-to-lotconsistency

EnvironmentalFactors

Invertebratecellculturesareextremelynsitivetoenvi-

-proteinnatureof

mostrum-freeformulationsoftenincreascellularnsi-

ceproblems,umaterialsandequipment

designatedfortissuecultureuonly,includingincubators,

flowhoods,autoclaves,mediapreparationareas,specialty

gas,theguidelineslistedhereto

ensurethatthephysicalconditionsofyourcultureoptimize

growth.

Temperature:Theoptimalrangeforgrowthandinfec-

tionofmostculturedinctcellsis25°Cto30°y

rum-supplementedmonolayerculturescanbestoredat

2°Cto8°Cforperiodsupto3months.

pH:ThepHofagrowthmediumaffectsbothcellular

proliferationandviralorrecombinantproteinproduction.

Althoughmanyvalueshavebeenreportedforinvertebrate

cells,inmostapplicationsapHrangeof6.0to6.4works

ectmedia

describedinthisguidewillmaintainapHinthisrangeunder

conditionsofnon-CO

2

equilibrationandopen-capped

culturesystems.

Osmolality:Theoptimalosmolalityofmediumforu

withlepidopterancelllinesis345to380mOsm/-

tainreliableandconsistentcellulargrowthpatternsand

minimizetechnicalproblems,maintainpHandosmolality

withintherangeslistedhere.

Aeration:Invertebratecellsrequiresufficienttransferof

dissolvedoxygenbyeitherpassiveoractivemethodsfor

optimalcellproliferationandexpressionofrecombinant

bioreactorsystemsusingactiveor

controlledoxygenationsystemsrequiredissolvedoxygenat

10%to50%ofairsaturation.

ShearForces:Suspensionculturetechniquesgenerate

sthatcontributetothetotal

shearstresxperiencedbycellsinsuspensionculture

includethesizeandtypeofimpellerswithinstirredvesls,

thesizeandvelocityofbubblesinairliftorspargedbio-reac-

tors,andtheresultingturbulentactionattheculturesurface.

Duringsuspensioncellculture,mostinctcelllinesrequire

ghrumconcentrations

between5%and20%inmediumappeartoprovidesome

protectionfromshearforces,werecommendthatall

suspensioncultures,whetherrum-freeorrum-supple-

mented,besupplementedwithashearforceprotectant

suchasPLURONIC®F-68.(Ifnotalreadyprentintheformu-

lation.)

5

GeneralMaterialsandEquipmentListThefollowingmaterialsandequipmentarerequiredto

onal,protocol-specificmate-

rialsarelistedwitheachprotocol.

•cellline(s)negativefortheprenceofmycoplasmaor

otheradventitiouscontaminatingagents(18,19)

•electroniccellcounter

•hemocytometerchamber

•incubatorcapableofmaintaining27°C±0.5°Candlarge

enoughtocontainthedesiredcultureconfiguration

apparatus

•invertedandphacontrastlightmicroscopes

•laminarflowhoodsuitableforcellculture

•low-speedcentrifuge

•pipetaide,automatedormanual

•pipets:1-,2-,5-,10-and25-mlvolumes

•37°Cwaterbath

•trypanblue

•completerum-supplementedorrum-freemediumof

choice

Protocol1:SubculturingMonolayerCultures

Note:Toensureadequateoxygenation,maintain

minimalmediadepthandloocaps.

MaterialsList

•Cellculture“T”-flasks,25-and/or75-cm2

teanddiscardthemediumandfloatingcellsfrom

an80%to90%confluentmonolayer.

25-cm2flask,add4to6mlofcompletegrowth

re

using75-cm2flasks,add15mlperflask.

endcellsbypipettingthemediumacrossthe

monolayerwithaPasteurpipette.

ethecellmonolayerusinganinvertedmicro-

scopetoensureadequatecelldetachmentfromthe

surfaceoftheflask.

inetheviablecellcountofharvestedcells(e.g.,

usingahemocytometerandtrypanbluedyeexclusion).

atecellsat2×104to5×104viablecells/cm2into

25-or75-cm2flasks.

teculturesat27°C±0.5°Cwithloocapsto

allowgaxchange.

turetheflaskswhenthemonolayerreaches80%

to100%confluency,approximately2to4dayspost-

gthoftimeneededtoreachconfluency

beforesubculturingoftendependsonthecellinocula

concentrationudinstep6.

Note:Ifthecelllineisgrowingslowly,feedtheflaskson

tespentmediumfrom

onesideofthemonolayerandgentlyre-feedwithfresh

turewhenmonolayerreaches80%to

100%confluency.

ProtocolNotes

•MasterCellSeedStock:Assoonasthecultureisfully

adaptedtothecultureconditionsandgrowthmedium,

prepareandcryoprerveamastercelledstock(e

Protocol5).Assomecelllinesmaybepassage-number

dependent,werecommendestablishingfreshcultures

periodically(e.g.,every3monthsor30passages)from

thefrozenmastercelledstock.

ForSerum-SupplementedCultures:

•AntibioticConcentrations:0.25µg/mlofamphotericin

B,100U/mlofpenicillin,and100µg/mlofstreptomycin.

•CareinHandling:Ucarewhenmovingrum-

ulturesdo

notadheretightlytomostglassorplasticsubstrates.

ForSerum-FreeCultures:

•AntibioticConcentrations:Antibioticsorantimycotics

antibioticsinrum-freeculture,reducethestandard

concentrations~50%.

•DislodgingtheCells:Inctcellsattachverytightlyto

substratesunderrum-freeconditionsandrequire

odgethecells,youmay

needtoshaketheflaskvigorouslytwotothreetimes

n:Toavoidcont-

amination,alwaystightenthecapbeforeshakingthe

flask.

Protocol2:AdaptingMonolayerCellsto

SuspensionCulture

Becauinctcellsarenotgenerallyanchorage

dependent,theyadapteasilytosuspensionculturecondi-

ectcelllinescommonlyudinBEVS

applicationshaveallbeensuccessfullyadaptedtosuspen-

sioncellcultures(eAppendixA).Itisimportantto

proceedslowlywhenadaptingstationaryculturesto

obrveadropinviabilityand

increadclumpingthroughthefirstthreetofivepassages.

Thisprotocolwilloptimizetheadaptationofmostinverte-

bratecelllinestosuspensioncultureandreduceor

10

confluent75-cm2monolayerflasksaresufficienttoinitiatea

100-mlsuspensionculture.

gecellsfromthebottomoftheflasks(e

Protocol1).

ecellsuspension,anddeterminetheviablecell

count.

thecellsuspensiontoapproximately5×105

viablecells/mlincompleterum-supplementedor

rum-freegrowthmediumequilibratedtoroom

temperature.

teat2.0°C±0.5°Cwithastirringrateof100rpm

forshakerflasksorastirringrateof75rpmforspinner

cultures.

turethecellswhentheviablecellcountreaches

1×106to2×106cells/ml(3to7dayspost-planting).

Increathestirringspeedby5to10rpmwitheach

6

olsforCulturingHostCells

7

viabilitiesdropbelow75%,

decreastirringspeedby5rpmforonepassageuntil

cultureviabilityrecoversandis>80%.

kerflaskcultures,repeatstep5untiltheconstant

stirringspeedreaches130to150rpm.

Forspinnercultures,repeatstep5untiltheconstantstir-

ringspeedis90to100rpm—unlessthespinnerflaskis

equippedwithamicro-carrierstirringasmbly(flat

bladeimpeller),inwhichcalimitmaximumstirring

speedto75to80rpm.

llshavefullyadaptedtosuspensionculture,

followProtocol3forroutinemaintenance.

ProtocolNotes

•Clumping:High-FiveBTI-TN-5B1-4andTn-368cell

linesoftendemonstrateavereclumpingproblemin

mizeclumping,

lettheculturesit2to3minbeforesubculturing,untilthe

largerclumps(>10cellsperclump)ttletothebottom

mplesforcountingandedingnew

culturesfromtheupperthirdofthesuspensionculture

(thistechniquelectsforacellpopulationthatgrowsas

singlecells).Ifnecessary,repeatthissteptwotothree

withveralrepetitions,5%to20%ofthecellpopulation

mayremaincompodofsmallclumps5to10cellsin

size.

•MasterCellSeedStock:Assoonasthecultureisfully

adaptedtothecultureconditionsandgrowthmedium,

prepareandcryoprerveamastercelledstock(e

Protocol5).Assomecelllinesmaybepassage-number

dependent,werecommendestablishingfreshcultures

periodically(e.g.,every3monthsor30passages)from

thefrozenmastercelledstock.

•Surfactants:Donotsupplementrum-freeinct

mediawithadditionalsurfactant,suchasPLURONICF-68.

Surfactantsareudinrum-supplementedculturesto

lesncellulardamageduetoshearforces,butconcen-

trations>0.10%maydecreagrowthorresultin

otherwi

indicated,mostSFMcontainsufficientsurfactant(s)to

protectcells.

•Magneticstirbarsdesignedtooperateonthebottomof

theflasksarenotsuitableforinctcellculture.

Protocol3:MaintainingSuspensionCultures

Thestandardflasksudinasuspensioncultureare

250-mldisposable,sterileErlenmeyerflasks(forvolumesof

50to125ml)and250-mlglassspinnerflasks(forvolumes

of150to175ml).Althoughyoucanscaleupshakerorspin-

nerflaskculturestoavarietyofveslsandvolumes,you

mustoptimizetherelativeflaskfillvolumesandstirring

le4fortypical

glassshakeorspinnerflasks,

besuretheflasksarethoroughlycleanedaftereachu.

Thisprotocolcanbeudwith250-mlshakeflasksor

spinnerflasks,alamountof

mediapercellsuspensionvolumeis50to125mlforshake

heconditions,

oxygentensionsarenotratelimitingandculturesachieve

maximumpopulationdoublingtimesanddensities.

mediumvolumes.

ShakerflaskSpinnerflask

Flasksize(ml)culturevolume(ml)culturevolume(ml)

12525–5050–100

25050–125150–200

500125–200200–300

1,000200–400300–1,000

3,000400–8002,000–3,000

MaterialsList

•disposableErlenmeyerflasks,125-,250-,and500-ml

•glassspinnerflasks,125-and250-ml

•orbitalshakerfittedfor50-to500-mlErlenmeyerflasks,

withshakingspeedofupto150rpm

•stirringplatformcapableofconstantoperationat90to

100rpm

•PLURONICF-68,10%(100X)

intheorbitalshakerorstirringplatformina

27°C±0.5°C,nonhumidified,non-CO

2

equilibrated,

culturesalreadyadaptedtoandmaintainedinsuspen-

sionculture,torbitalshakerat135to150rpmand

spinnerplatformsat90to100rpm.

a1-to2-mlsamplefroma3-to4-day-old

suspensionculture(inmid-exponentialgrowth)and

determinetheviablecellcount.

thecellsuspensionto3×105viablecells/mlin

completerum-freeorrum-supplementedgrowth

mediumequilibratedtoroomtemperature.

•Forrum-supplementedcultures:Youmayadd

10ml/LPLURONICF-68(0.05%to0.1%finalconcentra-

tion)tolesncellulardamagebyshearforces.

•Forshakerflasks:Maintainstockculturesasa50-to

100-mlculturein250-mlErlenmeyerflasks.

•Forspinnerflasks:Maintainstockculturesas150-to

175-mlculturesin250-mlspinnerflasks.

Fortypicalculturevolumes,tethe

cultures,loonthecapsabout¼to½ofaturn.

teculturesuntiltheyreach2×106to3×106

viablecells/tainconsistentandoptimalcell

growth,subculturesuspensionculturestwiceweekly.

ery3weeks,gentlycentrifugethecellsuspen-

sionat100×endthecellpelletin

freshmediumtoreducetheaccumulationofcelldebris

andmetabolicbyproducts.

ProtocolNotes

ForSpinnerCultures:

•Scalability:Thephysicalconstraintofproviding

adequateoxygentensionstotheculturelimitsthe

culture’evolumeinthespinner

veslbelow2/3fullandprovideforgasspargingasthe

veslsizeincreasabove500ml.

•CalibrationandAsmbly:Recalibratethegradation

marksoncommercialspinnerflasksusingagraduated

theimpellermechanismsrotatefreely

anddonotcontactveslwallorba.

turecellswhentheviablecellconcentration

reaches2×106to3×106cells/ml(about4to7days

post-planting).

ProtocolNotes

•Afterveralpassages,viablecellcountsofmostinct

linesshouldexceed2×106to4×106cells/ml.

Viabilitiesshouldbe>85%afterapproximately4to7

stage,thecultureisadaptedto

SFMandyoushouldcryoprerveamastercelled

stockforfutureu(eProtocol5).

Protocol5:PreparingaMasterCellSeed

Stock

Onceacultureisfullyadaptedtothecultureconditions

andgrowthmedium,itisntialthatyouestablisha

edstocks

shouldbepreparedusingthelowestpossiblepassage

oriesof25to50edstockampules(4-ml)

aregenerallysufficient;however,ifthemasterstockistobe

udforcGMPand/orlarge-scaleproduction,youmay

storeportionsofthe

mastercelledstockinmultiplefreezers,preferablyat

differentsites,toavoidthepossibilityofcatastrophicloss.

Withthisprotocol,youcancryoprerveupto504-mlvials.

MaterialsList

•automatedfreezer

•manualfreezertray

•cryovials

•appropriategrowthmedium(estep3)

siredquantityofcellsinsuspensionusingeither

tcellsinmid-logpha

ofgrowthwithaviability>90%.

inetheviablecellcount,andcalculatethe

requiredvolumeofcryoprervationmediumrequiredto

yieldafinalcelldensityof1×107to2×107cells/ml.

etherequiredvolumeofcryoprervation

medium.

Note:Forrum-freecultures,youhavetwochoices:

prepareamediumconsistingof7.5%DMSOin50%

freshSFMand50%conditionedmedium(sterile-

filtered),orprepareamediumconsistingof100%fresh

SFMcontaining10%BSAand7.5%DMSO.

Forrum-supplementedcultures,prepareafresh

mediumsupplementedwith7.5%DMSOand10%FBS.

hepreparedmediumandholdat4°Cuntilu.

fugecellsfromsuspensionormonolayerculture

mediumat100×thesupernatant.

Resuspendthecellpelletinthechilledcryoprervation

medium.

well-mixedaliquotsofcellsuspensioninto

cryovialsaccordingtovolumesrecommendedbythe

manufacturer.

eratecryovialsat0°Cto4°Cfor30min.

ervethevials,followingstandardprocedures

usingatemperaturereductionrateof1°Cperminute.

8

•Siliconization:Coatingculturewarewithanontoxicsili-

conizingagentmayminimizeattachmentofcelldebris

siliconized

themanu-

facturer’sguidelines,andtesttheefficacyof

siliconizationforyourprotocols.

ForSerum-FreeCultures:

•Dilutions:ForSf9andSf21cellsinSFM,donotdilute

thesuspensionculturesbelow3×105cells/o

Trichoplusiani(Tn-368orBTI-TN-5B1-4)cells,

eding2×105cells/mlissufficient.

Protocol4:AdaptingCulturestoSerum-Free

Medium

AdaptcellculturestoSFMsimultaneouslythroughboth

omaysaveyou

adaptingmonolayercellstoSFM,firstestablishthemto

suspensionculture(eProtocol2).Cellsmustbeinmid-

exponentialgrowthwithaviabilityofatleast90%.

Whenthecellsarecompletelyadaptedtorum-free

culture,theyshouldreachmaximumdensitiesandhave

populationdoublingtimescomparabletogrowthinrum-

supplementedmedium.

MaterialsList

•Sf-900IISFMorEXPRESS-FIVESFM

•Inctcellsadaptedtosuspensioncultureandgrowthin

rum-supplementedmedium

DirectAdaptationtoSFM:

culturescanbeadaptedtoSFMin5to8passages(~3

weeks).Ifviabilitiesdecreato<50%,orifculturesare

growingslowly(populationdoublingtimesare>72h)for

morethan3to4concutivepassages,uthequential

adaptationmethod.

mSFMto27°C±0.5°C.

ercellsgrowinginmediumcontaining5%to10%

FBSdirectlyintotheprewarmedSFMatadensityof5×

105cells/ml.

ecelldensityreaches2×106to3×106cells/ml

(4to7dayspost-eding),subculturethecellstoa

densityof5×105cells/ml.

turestockculturesofSFM-adaptedcells1to2

timesperweekwhentheviablecellcountreaches2×

106to3×106cells/mlwithatleast80%viability.

SequentialAdaptationtoSFM:

turecellsgrowninrum-containingmediuminto

a1:1ratioofSFMandtheoriginalrum-supplemented

mediawithaminimumedingdensityof5×105

cells/ml.

teculturesuntilviablecellcountexceeds

1×106cells/ml(aboutonepopulationdoubling).

Subculturecellsbymixingequalvolumesofconditioned

mediumandfreshSFM(1:1).

uetosubdividethecultureinthismanneruntilthe

rumconcentrationfallsbelow0.1%,cellviabilityis

>80%,andaviablecellconcentrationof>1×106

cells/mlisachieved.

Recovery:

Frozencellswillremainstableindefinitelyinliquidnitro-

iabilityofrecoveredcryoprervedcells24h

afterstoringvialsinliquidnitrogen,asfollows.

Caution:Forsafety,alwayswearafaceshieldwhen

o

willhelppreventinjuryifavialexplodesbecauofthe

rapidshiftintemperature.

mandequilibratecompletegrowthmedium.

rculturesfromfrozenstoragebyrapidly

thawingvialsina37°Cwaterbath.

sprayampuleexteriorwith70%ethanol.

ertheentirecontentsofthevialintoashakeror

spinnerflaskcontainingtheprewarmedmedium.

ateculturestoachieveaminimalviablecell

densityof3×105to5×105cells/ml.

intheculturebetween0.3×106and1×106

cells/mlfortwosubculturesafterrecovery,thenreturnto

thenormalmaintenanceschedule.

9

Severalmolecularbiologytechniquesareavailablefor

imal

results,followthemanufacturer’srecommendations

forbothhomologousrecombinationandsite-specifictrans-

positiontechniques.

PurifyingViralDNA

Severalfactorsarecriticalforhomologousrecombina-

ologousrecombination,pureviralDNAis

questopurifyviralDNAincludephenol

extraction(20),cesiumchloridepurification(20),oraffiinity

purificationwithamatrixsuchasCONCERT

™HighPurity

iceof

protocoldependsontheamountofwild-typebaculovirus

DNAneeded.

Protocol6:IsolationofBacmidDNAforBAC-

TO-BAC®BaculovirusExpressionSystemwith

theCONCERTHighPurityPlasmidPurification

System

WehaveisolatedbacmidDNAfromDH10BACwiththe

CONCERT™HighPurityPlasmidMiniprepsystemusingthe

~150kbbacmid(GUScontrol)was

Awas

ere

harvestedat48hand72hpost-transfectionandstained

encieswere

similartothoobrvedwithtransfectionsusingbacmid

DNAisolatedbyothermethods.

InoculationofwhitecolonyintominiprepLB

kan,gent,tetbrothculture:

Inoculateasingle,whitebacterialcolonyinto2mlofLB

kan,gent,tetbroth(Falcon®2059tube.)Placethebroth

cultureintheshakingwaterbathat37°Cand250rpmfora

minimumof16hours(overnightisfine.)

IsolationofrecombinantbacmidDNA:

beginning:Verifythatnoprecipitatehas

formedinCellLysisSolution(E2.)IfthesolutionE2istoo

cold,:Make

sureyouhaveaddedRNaAtoCellSuspensionBuffer

(E1.)

Equilibration:Apply2mlofEquilibration

Buffer(E4)[600mMNaCl,100mMsodiumacetate(pH

5.0),0.15%TritonX-100]hesolution

inthecolumntodrainbygravityflow.

rvesting:Pellet1.5mlofanovernightculture.

Thoroughlyremoveallmedium.

spension:Add0.4mlofCellSuspension

Buffer(E1)[50mMTris-HCl(pH8.0),10mMEDTA,contain-

ingRNaAat0.2mg/ml]tothepelletandsuspendcells

untilhomogeneous.

10

sis:Add0.4mlofCellLysisSolution(E2)[200

mMNaOH,1%SDS].Mixgentlybyinvertingthecapped

teatroomtempera-

turefor5min.

lization:Add0.4mlofNeutralizationBuffer

(E3)[3.1Mpotassiumacetate(pH5.5)]andmiximmedi-

ortex.

Centrifugethemixtureattopspeedinamicrocentrifugeat

entrifugeat4°C.

Loading:Pipetthesupernatantfromstep12

hesolutioninthe

dflow-through.

Wash:Washthecolumntwotimeswith2.5

mlofWashBuffer(E5)[800mMNaCl,100mMSodium

acetate(pH5.0)].Allowthesolutioninthecolumntodrain

dflow-through.

dDNAElution:ElutetheDNAbyadding0.9ml

ofElutionBuffer(E6)[1.25MNaCl,100mMTris-HCl(pH

8.5)].Allowthesolutioninthecolumntodrainbygravity

orceoutremainingsolution.

dDNAPrecipitation:Add0.63mlof

placeonicefor10min.

Centrifugethemixtureattopspeedinamicrocentrifugeat

llydiscardsuper-

eplasmidDNApelletwith1mloficecold

70%llyandfully

thepelletfor10min.

edDNA:DissolvethepelletedDNAin40µlof

TEBuffer(TE)[10mMTris-HCl(pH8.0),0.1mMEDTA].

d

DNAshearing,pipetDNAonly1-2timesduringresuspen-

sion.

BacmidDNAcanbestoredat-20°C,butavoidrepeated

freeze/thawing.

U5µlofthisbacmidpreparationfortransfectionof

inctcells.

PreparationofMedia:

LuriaAgarPlates:Miller'sFormulation(Premixed

formulationofMiller'sLBPlatesisavailable:.12945-

036)

Note:UofLennoxL(LB)agarinsteadofMiller's

formulationLuriaagarplateswillreducecolorintensityand

ofX-galinstead

ofBluo-galwilldecreacolorintensity.

olsforGeneratingaRecombinantBaculovirus

ComponentAmount

SELECTPeptone14010g

SELECTYeastExtract5g

sodiumchloride10g

SELECTAgar12g

distilledwatertoavolumeof1L

lutionto55°oticsand

supplementsareaddedtothecooledsolution.

Component

onc.

kanamycin10mg/ml(indistilledwater)50µg/ml

gentamicin10mg/ml(indistilledwater)7µg/ml

tetracycline5mg/ml(inethanol/pH-titrated)10µg/ml

IPTG200mg/ml(indistilledwater)40µg/ml

Bluo-Gal20mg/ml(inDMSO)300µg/ml

t-20°Cas

agarsolutionpriortopouring25mlper

gar

platesinvertedinplasticat4°Cforuptofourweeksinthe

dark.

Protocol7:CationicLiposome-Mediated

TransfectionUsingCellFECTIN™Reagent

DNAcanbetransfectedintoinctcellsusingcalcium

phosphatecoprecipitation,DEAE-dextran-mediatedtrans-

fection,liposome-mediatedtransfection,electroporation,

tooptimizeconditionsfor

hestefficiencyhasbeenachievedwith

CELLFECTINReagent.

Fortransfectiontobeefficient,youmustuhighlypuri-

fywild-typeviral

DNA,youmayuapublishedprocedureorProtocol6.

ThisprotocolhasbeenoptimizedforSf9cellsgrownin

CTINReagentcanbeudforcellsgrownin

rum-containingmediumaslongasyouformthelipid/DNA

complexesintheabnceofrum.

MaterialsList

•steriletubes,12×75-mm

•tissuecultureplate(s),6-well

•CELLFECTINReagent

•0.5Xpenicillin/streptomycin/neomycin

•Sf9orBTI-TN-5B1-4cells,growingexponentiallyata

minimumconcentrationof5×105viablecells/ml

•Sf-900IISFMorEXPRESS-FIVESFM

6-welltissuecultureplate,ed9×105Sf9cellsper

wellin2mlofSf-900IISFMor9×105BTI-TN-5B1-4

cellsperwellin2mlofEXPRESS-FIVESFM(withantibi-

otics).

tetheplateat28°Cforatleast1htoallowcells

toattach.

12×75-mmsteriletubes,preparethefollowing

solutions.

SolutionA:Foreachtransfection,dilute1to2µg

baculovirusDNAand5µgtransfervectorofchoice

into100µlSf-900IISFMorEXPRESS-FIVESFM

withoutantibiotics.

11

SolutionB:Foreachtransfection,dilute1.5to9µl

CELLFECTINReagentinto100µlSf-900IISFMor

EXPRESS-FIVESFMwithoutantibiotics.

utionBtothetubecontainingSolutionA,mix

gently,andincubateatroomtemperaturefor15min.

ipid/DNAcomplexesareforming,washtheSf9

cellsfromstep2oncewith2mlperwellofSf-900IISFM

withoutantibiotics.

0.8mlSf-900IISFMtoeachtubecontaining

lipid/tethewash

medium,andoverlaythedilutedlipid/DNAcomplexes

ontothewashedcells.

tefor5hina27°Cincubator.

2mlSf-900II

SFMorEXPRESS-FIVESFM(containingantibiotics)per

wellordishandincubateat27°Cfor72h.

tthevirusfromthecellculturemediumat72h

post-transfection.

Protocol8:VirusPlaqueAssay

Theinfectiouspotencyofastockofbaculovirusisdeter-

minedbyexaminingandcountingplaqueformationsinan

ngtechniquesare

5isprovidedasatroubleshootingguideforthisprotocol.

Manyvariationsofthebasictechniqueareud,andeach

providessomeadvantagesdependinguponthecellline

employed,natureoftherecombinantconstruct,andidentifi-

cation/otocolcanbe

adaptedtoaccommodatevariations.

MaterialsList

•cellcultureplates,6-well

•centrifugetubes,12-mlpolystyrene(disposable)

•glassbottle,100-mlsterile(empty)

•Pasteurpipet,sterile,plugged

•sterilepipets,one1-mlandone10-ml

•70°Cwaterbath

•4%agarogelor4%agarogelwithBluo-gal

•baculovirussupernatant,clarified,cell-free,sterile

•distilledwater(sterile),cell-culture-grade

•exponentialcultureofSf9,Sf21,orBTI-5B1-4cellsat5

×105cells/ml

•inctcellculturemedium:Sf-900(1.3X)orGrace’s

InctPlaquingMedium(2X)plusheat-inactivatedFBS.

Note:Forplaquing,Sf900(1.3X)canbeudifcellsare

growninanySFM.

terileconditions,dispen2mlofcellsuspen-

sion(5×105cells/ml)intoeachwell.

te,

covered,grum-

supplementedmedia,transporttheplatesgently

becaucellsdonotadheretightlytotheplatesurface.

hebottleofagarogelinthe70°Cwaterbath.

Placetheempty100-mlbottleandthebottleof1.3X

Sf-900InctMedium(or2XGrace’sInctMedium)in

the37°Cwaterbath.

differentscreeningmethodsareappropriatefordifferent

culoviralplaquesfitoneofthefollow-

ingfourcategories:

-type:Plaquesfromwild-typeAcMNPVinfections

inagarooverlaystendtobehighlyrefractileandnear-

quescanbeidentified

llappearas

regionsofdecreadcelldensitycontainingmanycells

leiwillcontainmanylarge,

dark,angularocclusionbodies.

inant:Plaquesfromrecombinantvirusinfec-

tions(i.e.,ofco-transfectedconstructs)canbedifficultto

ky-greyplaquesaresmall,oflow

contrast,especiallytrue

whentheyreprentasmallpercentageofthetotal

lobliqueilluminationbyahigh-

intensitylightsourcecanrevealcandidatesforquantita-

gorscoringthecandidateswithafelt-tipped

lowingmethodsare

ufulforidentifyingplaquesfromrecombinantvirus

infections:

•Stainingwithneutralredsolution(Protocol13)or

MTT(0.5mlofa1mg/mlsolutionperwell).Score

thewild-typeplaquesthenstaintoidentifyunscored

recombinantplaquesafterstaining.

•Southernblothybridizationofbuddedvirusfromthe

vicinityofaplaquecanconfirmtheprenceofthe

eans(e.g.,Westernblotor

functionalassay)arenecessarytoestablishthe

cloneasasuccessfulproducerofprotein.

inantxpressingchromogenicmarkers:If

therecombinantvirusbearsareportergenethat

producesvisiblecolorimetricreactions,plaquescanbe

detected,counted,

uavectorthatcontainsluciferaorβ-galactosida

tohelprevealtheminority(0.1%to3%)ofsuccessful

genicmarkersalsomakeiteasier

-galand

X-galrevealrecombinantplaquexpressingthelacZ

geneproductbyproducingadeepblueprecipitate.

inantsproducingproductsthatcanbe

monitoredimmunologically:Theproductsare

distinguishedbyWesternblotting.

12

he1-hincubation,obrvemonolayersunderthe

invertedmicroscopetoconfirmcellattachmentand50%

confluence.

ea10

-1

to10

-8

rialdilutionoftheharvested

viralsupernatantbyquentiallydiluting0.5mlofthe

previousdilutionin4.5mlofSf-900IISFM(orGrace’s

InctCellCultureMedium,Supplemented,without

FBS)uldhave

eighttubescontaining5mleachoftherialdilution

fromeachoriginalvirusstock.

e6-wellplatesandthetubesofdilutedvirusto

achdilutioninduplicate.

tiallyremovethesupernatantfromeachwell,

discard,andimmediatelyreplacewith1mlofthe

tefor1hatroom

temperature.

eoneofthefollowingplaquingoverlays:

•Sf-900plaquingoverlay:Movebottlesfromwater

baths(fromstep3)toasterilehoodwhentheagaro

hasliquified(after20to30min).Quicklydispen30

mlofthe1.3XSf-900InctPlaquingMediumand10

mlofthe4%

thebottleofplaquingoverlaytothe

37°Cwaterbathuntilu.

•Grace’splaquingoverlay:Movebottlesfromwater

baths(fromstep3)toasterilehoodwhentheagaro

hasliquified(after20to30min).Apticallyadd20ml

ofheat-inactivatedFBStotheGrace’sInct

e25mlofthe

Grace’sInctMediumsupplementedwithFBS,

12.5mlofcell-culture-gradesterilewater,and12.5ml

ofthemelted4%AgaroGelintothe

theplaquing

overlaytothe37°Cwaterbathuntilu.

he1-hincubationwithvirus,returnthebottleof

dilutedagaroandthe6-wellplatestothehood.

tially(fromhightolowdilution)removethevirus

inoculumfromthewellsandreplacewith2mlofthe

icklytoavoiddesiccationofthe

urpipetconnectedtoa

vacuumpumpeasilyremovesinoculumtraces.

gelharden10to20minbeforemovingthe

plates.

tetheplatesat27°Cinahumidifiedincubatorfor

4to10days.

rplatesdailyuntilthenumberofplaquescounted

doesnotchangefor2concutivedays.

ProtocolNotes

•Titer:Todeterminethetiteroftheinoculumemployed,

malrangeto

countisbetween3and20plaquesperwellofa6-well

calculatethetiterinplaque-forming

units/mlusingthefollowingformula:

pfu/mloforiginalstock=1/dilutionfactor×numberof

plaques×1/(mlofinoculum/plate)

Identifyingtheplaques

Becauplaquesareidentifiedbytheirphenotype,

13

eshootingvirusplaqueassays.

ProblemPossibleCauSolution

NoorsmallplaquesPhysicalconditionofcellsispoorUcellsinmid-logphagrowthwithviabilities>90%.

(otherparametersappearfine)

CelledingdensitytoohighDecreaedingdensityto106cellsperwellina6-wellplate

(40%to50%confluency).

InhibitionofviralreplicationcycledueBesuretomakeagarooverlaywith1.3XSf-900or

toinadequatenutrition,temperature,or2XGrace’sMedia.

atmosphericconditions

MisdilutionorinactiveinoculumMaintainplatesat27°Cinanon-CO

2

atmosphere.

Note:IftherecombinantviruscontainsCheckthatthedilutionsweredoneproperly.

acytotoxicexogenousgeneproductor

inhibitsbuddedvirusproduction,the

resultisnoplaques.

SmallplaquesToomanyplaquesontheplateInoculateatahigherdilution.

PrematuredeathofthemonolayerdueIncreahumidityintheincubator(e.g.,putplatesintoa

todesiccationoftheoverlaycontainerwithadampcloth).

Moveplatesawayfromwallofincubator.

Increavolumeofoverlay.

PlasticwaremayaffectinctcellEvaluateadifferentstyleorvendorofplasticware.

attachmentandgrowth

LargeplaquesCelledingdensitytoolowIncreaedingdensityto106cellsperwellina6-wellplate

(hardtoidentify)(40%to50%confluency).

InhibitionofcellgrowthduetoBesuremediumisaddedtotheagarooverlay.

inadequatenutrition,temperature,Maintainplatesat27°Cinanon-CO

2

atmosphere.

oratmosphericconditions

InadequateimmobilizationoftheBesuretocompletelyremovetheinoculum.

monolayer

PoorgellingoftheoverlayU4%agarostockanddilutewithmediumto2%.

DrippingofcondendmoisturedownAllowplatestocoolwithlidsopenafteraddingagarooverlay.

thewallsofdishes

GelisdetachedfromthesurfaceoftheDonotshakeplatesafteroverlayisgelled.

monolayer

Crescent-shapedpatchesMonolayerdriedpartiallybeforeadditionKeepcellsmoistthroughouttheentireprocedure.

ofeithertheviralinoculumorgeloverlay

UnevenformationofthemonolayerAllowcellstoattachonanevensurface.

NoplaquesorsmallerplaquesCellinoculumwasdistributedbyDistributeinoculumbyrockingtheplate.

inthecenteroftheplatewith“swirling”

larger“smeared”plaquesin

peripheralregionsoftheplate

Blueregionsofβ-galactosidaToomuchchromogenicsubstrateinUafinalconcentrationof300µg/mlBluo-gal.

expressiontoolargeoverlay

PlaquesoverdevelopedDevelopplatesfor3daysandscoreplaquesdailyuntilplaques

aredistinct.

DiffusionofdyewithingelUBluo-galtominimizediffusion.

NearlyinvisiblerecombinantObrvationforsomehomologousDevelopplates(3to7days)atroomtemperaturetoincreathecontrast

plaqueswhilewild-typerecombinationmethodsinrecombinantplaques.

plaquesarequitedistinctUacolorimetricmarkerinthetransferplasmid.

StainthemonolayerwithneutralredorMTT.

BubblesonsurfaceBubblesintroducedintothemoltenDrawup1mlmoreagarothantheprocedurerequiresanddonot

ofagarooverlayagaroexpelentirecontentsfortheoverlay.

Touchbubbleswithheatedsterilepipetorbrieflyflamesurfacetopop

bubbles.

Protocol10:AmplifyingtheVirusStock

Beforeyouamplifyorexpandthevirusstock,itisn-

tialthatyouknowthetiterofyourtransfectionsupernatants

nMOIof<0.50will

preventbuildupofdefective,interferingvirusparticles.

Defective,interferingvirusparticlebuildupisaconcern

particularlyaftermultipleviruspassagesandforvirus

owMOI,cell

entrate-

limitingnutritionalproblemsthatmayresultindecread

viralproductionandtitersduringexpansionofvirusstocks,

followtheguidelinesformaximumviablecelldensitiesin

table6.

asuspensionormonolayercultureinmid-

exponentialgrowthatanMOIof0.01to0.1accordingto

thefollowingformula:

Inoculumrequired(ml)=

DesiredMOI(pfu/cell)×(totalnumberofcells)

Titerofviralinoculum(pfu/ml)

Note:At48hpost-infectionusuallyyieldsa2-logampli-

fication.

Example:Infecta50-mlcultureofSf9cellsat2×106

cells/mlwith0.5mlofaviralstockcontaining2×107

pfu/mltoobtainanMOIof0.10.

he

virusstockbyplaqueassay(eProtocol8).

steps1and2untilvirusstockhasaconfirmed

titerof1×107to1×108pfu/ml.

hevirusstocksat4°Cforupto1year,protected

fromlight(eProtocol15).

Protocol11:IdentifyingPlaquesbyNeutral

RedStaining

Incubationtimesandtheamountofstainudvary

the

platesgentlythroughoutanystainingprocedureasthe

monolayeriasilydisrupted.

14

Protocol9:PlaquePurificationof

RecombinantViralClonesForhomologousrecombination,threeroundsofplaque

purificationwillensuregenerationofapurerecombi-

purificationisnotnecessary

withthesite-specifictranspositionmethod.

MaterialsList

•alableplasticcontainer(~4×8×8in.)

•tissuecultureplates,6-well

•plateswithwell-developedOcc(-)plaques

•Sf9orBTI-5B1-4cells,growingexponentially(viability

>95%),ataminimumconcentrationof5×105viable

cells/ml

•completerum-freeorrum-supplementedinct

mediumofchoice

chwellwith2mlSf9orBTI-5B1-4cell

suspension,at5×105viablecells/mlinfreshmedium.

eplatescontainingplaquesbelowputative

istanceinidentifyingrecombi-

nants,eIdentifyingthePlaques.

terileconditions,removeplugsoftheoverlay

eroneplugtoeach

wellofamulti-wellplate.

tetheplateinahumidifiedchamberat27°C.

ethewellsdailyforsignsofinfectionand

abnceofpolyhedra.

4or5,point,you

mayscreenandconfirmthattherecombinantvirus

areproducingthegeneofinterest.

ingProtocol8,replaque10

-1

to10

-3

dilutionsof

:Itisnotnecessarytoprepare

thefullrange(10

-1

to10

-8

)the

plaquepurificationoftherecombinantvirustwiceand

determinevirustiters.

yconfirmedpurifiedproducersineithermonolayer

orshakerinfectionsatamultiplicityofinfection(MOI)of

tocksat

4°Cforupto1year,protectedfromlight(eProtocol

15).

olsforPurifyingandProducingRecombinantAcNPV

andProtein

endedmaximuminfectiondensitiesfortheproductionofrAcNPVorrecombinantproducts

Serum-freemediaSerum-supplementedmedia

MonolayerSuspensionMonolayerSuspension

cultureculturecultureculture

.

linerAcNPVproductrAcNPVproductrAcNPVproductrAcNPVproduct

Sf-90.50–1.00.50–1.0*1.5–2.02.0–3.00.50.51.0–1.51.0–2.0

Sf-210.50–1.00.50–1.01.0–1.51.0–2.00.50.51.01.0–1.5

Tn-3680.25–0.500.25–0.500.5–1.51.0–2.00.25–0.50.25–0.500.5–1.01.0

BTI-TN-5B4-10.25–0.500.25–0.500.5–1.51.5–2.0NANANANA

*Numbersareviablecells/ml×106.

MaterialsList

•distilledwater,cell-culture-tested

•neutralredstainingsolution(3.3g/L)

•plateswithdevelopedOcc

-

plaques

quepurification,scoreallvisibleplaqueswitha

llmakeiteasiertoidentifypotential

producersofrecombinantproduct.

ypreparea0.1%(w/v)neutralredstainsolutionin

cell-culture-gradewater.

wellcontaining2mlofplaquingoverlay,add0.5

mlof0.1%tefor1to2hat

roomtemperature.

removeexcessstainwithapipetorblotter.

yieldclearplaquesinanearlycleargelagainsta

edplaquesmadevisibleby

stainingarepotentialrecombinants.

Protocol12:OptimizingVirusStockProduction

Thisprotocolcanbeudtooptimizeandproducehigh-

quality,high-titermasterorworkingvirusstocks.

MaterialsList

•completerum-freeorrum-supplementedmediumof

choice

•high-titerrAcNPVstock(>1×107pfu/ml);and

•shakeorspinnerflasks

ndinoculate15replicaterum-freeorrum-

supplementedsuspensionculturesintriplicateas

describedinProtocol3.

lturesfor2to3daysuntiltheyareinmid-

exponentialgrowth(16-to24-hdoublingtimes)and

haveattainedthecelldensitiesrecommendedfor

infectionintable6.

Note:Ifthecellcultureexceedsthedensityrecom-

mendedintable5,dilutethecellculturebeforeinfection

withupto50%,however,thatthe

totalvolumedoesnotexceedthatrecommendedintable

4.

triplicateflasksateachofthefollowingMOIs:

0.01,0.05,0.10,and0.50(eProtocol10,step1,to

determinevirusinocularequiredateachMOI).Maintain

onetofflasksasuninfectedgrowthcontrols.

flasks24,48,e

morphologiesandcelldensitiesofinfectedcultures

againstnoninfectedcontrolstoconfirmprogressof

inetotalandviablecellcountsand

store1to5mlofclarified,sterilevirusfromeachsample

at4°C.

inethevirustiterofeachsamplebyplaque

assay(Protocol8).

theoptimalMOIandtheharvesttimethat

producedthehighestcombinationofvirustiterand

cultureviability>80%.Producealargequantityofwork-

ingand/ormastervirusstockusingtheinfection

parameters.

orkingvirusstocksat4°Candmastervirus

stocksat–70°Corinliquidnitrogen,asrecommendedin

Protocol15.

15

Protocol13:HarvestingtheVirus

Extracellularvirus,orbuddedvirus,beginsaccumulating

inthegrowthmedium~8to10hpost-infectionandcontin-

uesaccumulatingthrough~ynchronized

infection(MOI>4.0),buddedvirusproductioniscompleteat

~slittleornobenefittolonger

viruswithfunctionaltitersispossibleat

tingbeforethelyticphawhen

thecellviabilitiesare>90%willminimizecontaminationby

celldebris,metabolicwasteproducts,

non-synchronousinfections(MOI<4.0),buddedviruscan

beharvestedthroughapproximately48hpost-infection.

Withthisprotocol,lossofvirustiterwillbeminimal

(<10%).Furtherpurificationofthevirusisnotusually

necessary.

MaterialsList

•centrifugetubes

•0.2-µmlow-proteinbindingfilterunit

oraspiratethegrowthmediumcontainingvirus

fromthecultureintocentrifugetubes.

fugeat250×gfor5mintoremovecellsandlarge

debris.

Forsuspensioncultures:Ifdesired,centrifugeacond

timeat1,000×gfor20to30min.

efilter,ifdesired,througha0.2-µmlow-protein

bindingfilter.

irusasrecommendedinProtocol15.

Protocol14:ConcentratingtheVirus

ToproduceviralDNAortoachieveanotherwiunob-

tainableMOI(>10.0),uthisprotocoltoconcentratethe

ernatantmustbe

harvestedfromanonlytic,rum-freeculture.

MaterialsList

•ultracentrifugetubes,38-mlpolyallomer

•0.2-µmlow-proteinbindingfilterunit

•virusstocktobeconcentrated

•sucrosolution:25%sucroin5mMNaCl,10mM

EDTA

•Dulbecco’sPhosphate-BufferedSaline(D-PBS)(pH6.2)

33mlofvirusstockintoeachofsix38-mlpolyal-

lomerultracentrifugetubes.

aythevirusstockwith3mlofsucrosolution

pertube.

fugeat80,000×gfor75minat4°C.

thesupernatant,removingasmuchfromthe

ivelypureviral

pelletwillbetranslucentwhite,withfaintbluecolornear

repelletsdisplayincreasingopaque-

nessandsize;theircolorrangesfrompale

yellowtolightbrownascontaminationincreas.

endpelletsin0.5to5mlD-PBSorcellgrowth

sufficienttimeafterresuspensionforthecellstodisrupt

througha0.2-µt4°C.

importanttodeterminetheexpressionkineticsforeach

product,asmanyproteins(cretedornoncreted)may

bedegradedbycellularproteasreleadincellculture.

Toexpresssomerecombinantproductsand/orrAcNPV,

youmayneedtoprotecttherecombinantproductorvirus

fromproteolysisbysupplementingrum-freeculturespost-

infectionwith0.1%to0.5%n-bad

proteainhibitorsaregenerallylesxpensiveandmore

effectivethanmanysyntheticproteainhibitors.

Thisprotocolissuitablefordeterminingboththeoptimal

MOIandharvesttimefortheproductionofyourrecombinant

product.

MaterialsList

•completerum-freeorrum-supplementedmediumof

choice

•high-titerrAcNPVstock(>1×107pfu/ml)

•shakeorspinnerflasks

ndinoculate15replicaterum-freeorrum-

supplementedsuspensionculturesasdescribedin

Protocol3.

lturesfor2to3daysuntiltheyareinmid-expo-

nentialgrowth(16-to24-hdoublingtimes)andhave

attainedthecelldensitiesrecommendedforinfectionin

table6.

Note:Ifthecellcultureexceedsthedensityrecom-

mendedintable5,dilutethecellculturebeforeinfection

withupto50%,however,thatthe

totalvolumedoesnotexceedthatrecommendedintable

4.

triplicateflasksateachofthefollowingMOIs:

0.50,1.0,5.0,and10.0(eProtocol10,step1,to

determinevirusinocularequiredateachMOI).Maintain

onetofflasksasuninfectedgrowthcontrols.

flasks24,48,72,and96hpost-infection.

Comparemorphologiesandcelldensitiesofinfected

culturesagainstnon-infectedcontrolstoconfirm

inetotalandviablecell

counts.

Note:Optimalproductexpressionisoftenbetween

48and72hpost-infection,soyoumaywanttosample

culturevery8to12hafter24hpost-infection.

ellpelletfrom1to5mlofcellsuspension

at-20°C(fornoncretedproducts)or1to5mlofclari-

fiedsupernatantsat4°C(forcretedproducts).

ellpelletsorsupernatantsamplesforrecombi-

nantproductyieldsand/oractivity.

theoptimalMOIandtheharvesttimethat

producedthehighestcombinationofproductyield/activ-

ityandquality/homogeneity.

ptheproductionofrecombinantproductusing

irmoptimalharvest

timeafterscale-up.

Protocol15:StoringtheVirus

Virionsarequitestableinstandardrum-supplemented

intaintheirintegrityandinfectious

competencyfordaysatelevatedtemperatures,weeksat

roomtemperature,andmonthstoyearsat4°C.

Ifvirionswillbestoredforlongerthan3monthsunder

rum-freeconditions,add0.1%to1%BSAtostabilizethe

hevirusstocksinpolypropylenecontainersor

siliconizedglasswaretopreventnonspecificbindingof

ouldberetiteredperiodicallyifudasinocu-

virustiterwillbeminimal(<10%)withthis

protocol.

MaterialsList

•centrifugetubes

•sterilecryotubes(orotherlarge-volumecontainer

suitableforfreezing)

•0.2-µmlow-proteinbindingfilterunit(optional)

•virussupernatant

callytransfervirus-containingsupernatanttoa

sterile,fuge5minat

500×ortransferthevirus-containingsuper-

natanttoafreshtube(s).

efilter,ifdesired,througha0.2-µm,low-protein

bindingfilter.

theclarified,sterile-filteredsupernatantinto

cryotubes(orsuitablelargervolumecontainers).

hevirusstocksat4°C,

long-termstorageat4°C,–70°C,orinliquidnitrogen,we

recommendaddingBSAtoafinalconcentrationof0.1%

to1%.

Protocol16:OptimizingHeterologousProtein

Production

Thefirststeptowardsuccessfulinfectionofinctcells

witheitherwild-typeorrecombinantbaculovirusinsuring

thattheculturewillnotberatelimitedbynutritionalfactors

(i.e.,aminoacidorcarbohydrateutilization)orenvironmen-

talfactors(i.e.,pH,dissolvedO

2

,temperature).Cultures

shouldbeinfectedwhileinthemid-logarithmicphaof

imalMOIvariesby

celllineandtherelativeinfectionkineticsofthevirusisolate

espon(orMOI)shouldbe

establishedforeachvirus,medium,reactor,andcellline

formationwillenableyoutodetermine

optimalinfectionparametersforproductionofvirusor

recombinantproduct.

Whenproducingnon-occludedvirusstock(recombinant

orwild-type),infectthesuspensioncultureatacelldensity

of1×106to2×106cells/mlwithanMOIof0.01to0.1(e

Protocol12andtable5).Toexpressrecombinantgene

products,MOIsof0.5to10arecommonlyemployed.

Standardrum-supplementedmediaudforvirusinfec-

tionareratelimitingifthecellsareinfectedatdensities

>2×106cells/r,withSf-900IISFM,suspension

cultureshavebeensuccessfullyinfectedat

2×106to3×106cells/ml,andsuccesshavebeen

reportedat>4×106cells/ml(21).

TheBEVSrecombinantgeneproductmayormaynotbe

mexpressionisusuallyobrved

between30and72hforcretedproteinsandbetween

16

17

ingRecombinantProteins

Thefollowingcriteriaareimportanttoconsiderwhen

lectingapurificationprotocol:

•ScaleofExpression:Protocolfficientinsmallscale

maynotbeefficientinlargescale.

•NatureoftheProductExpresd:Considerusing

immunoaffinitychromatographywhenalow-costsource

ofpureantibodyexistsfortheprotein.

•GrowthMedium:Serum-freeculturesupernatants

harvestedfrominfectedculturesbeforesignificantcell

lysisoccursmayhaverecombinantproductasamajor-

ity(upwardsof95%)ofthetotalproteincomplement.

•ProductApplication:Practicaland/orregulatory

demandsmaydeterminethepurificationapproach.

Whendesigningapurificationprotocol,considerthe

impactofeachofthefollowing:

UofHydrolysates,Extracts,Lipids,andSterols:

canhavesomeunpredictableinteractionswithboththe

proteinofinterestand/orthechromatographictechnique.

Affinitychromatographygenerallywilleliminateproblems

annotuaffinity

chromatography,trytoeliminatethemediacomponents

inthefirstpurificationstep(i.e.,diafiltrationwithabuffer

exchangestep).

UofPLURONICF-68Co-polymer:Mostrum-free

inctcellculturemediacontainsurfaceactiveagentssuch

asPLURONICF-68thatcancauproblemsduringcertain

ICF-68mayexistinculture

asawiderangeofpolymericstructuresdependentupon

concentration;pH;temperature;andtheprenceofother

surfactant(s),detergents,lipids,sterols,orpolarmolecules.

AlthoughPLURONICF-68doesnotinterferewithmanychro-

matographicandprecipitationtechniques,itwill

precipitateintheprenceofhighsaltconcentrations.

Beforefurtherprocessingthatmayinvolvehighsalt

concentrates,suchas(NH

4

)

2

SO

4

precipitationorhydro-

phobicinteractionchromatography(HIC),diafiltratewitha

bufferexchangestep.

PrenceofaCystineProtea:Ambientmediumof

baculovirusinfectedcellsmaycontainacystineprotea

(22,23).Proteolysisisariousissueinrum-free

eSFMarelowinproteinorprotein-free,

theyprovidelittlecompetitivesubstratefortheprotea

edproteinshavedemonstratedavariable

chershaveexam-

inedavarietyofproteainhibitorswithvariablesuccess.A

reportusingpCMBS(p-chloromercuribenzene)appears

promising(24).Thebestwaytoreducethechanceofsignif-

icantproteolysisistokeeppost-infectionculture

supernatantsrefrigerated,toharvesttheproductbefore

significantcelllysisoccurs,andtoprocesstheproductas

onof0.1to1%BSA

canprovideacompetitivesubstratefortheprotea.

SecretedProteins:Proteinxpresdinthe

baculoviruxpressionvectorsystemaccumulateextracel-

lularlyinthegrowthmediumascretedproteins,or

tproteinswithabntoraberrant

signalquencesmaynotprocessnormallyand,asa

result,olsforthepurificationof

intracellularproductbeginwiththephysicalorchemical

disruptionofcells,followedbyisolationprocedures.

Toclarifycretedproteins,uttling,centrifugation,

rprocessingofthesupernatantcan

includegelfiltration,chromatography,andprecipitation.

PurificationfromSf-900IISFMorEXPRESS-FIVESFM

ThechiefadvantagetousingSFMforcultureofinct

cellsisthatpurificationprotocolsaresimplifiedbecau

advantageis

thepossibleproteolyticdegradationofproteinswhen

concentratingproduct.

PurifyingSecretedProteins

Uthefollowingguidelinestopurifycretedproteins.

Tosimplifypurificationprotocolsandpreventproblemsin

latersteps,werecommendathoroughbufferexchangeor

washingearlyinthepurificationsuchasattheconcentra-

tionstep.

RemovingCells

Supernatantsshouldbeclarifiedbeforefurther

processing.

Forsmall-scalecultures:

Centrifugationfor5minat1,000×gmaybesufficient.

Youcanalsoremovethevirusbyultracentrifugationat

80,000×gfor75min.

Forlargeliquidvolumes:

Youhaveveraloptionsforremovingcellsinlarge

clarifythesupernatantwith

antageofcartridge

canuultrafiltrationmembranes,butthetendtofoul.

Forcross-flow,tangential-flowandhollow-fiber

systems,youcanumicroporousfiltermembranes.

Theofferahigherfluxrateandarelesslikelytofoul.

RemovingBaculovirus

Optionsforremovingbaculovirusfromsmall-orlarge-

scaleculturesupernatantsincludemembranefiltration

apparatusandchromatographictechniquessuchasanion

einformationonvirusremovalandinac-

tivation,eGrunetal.(25).

ConcentratingtheProduct

Theproductcanbeconcentratedbydialysis,membrane

filtration,l-

ysisandmembranefiltration,uamembranewitha

10-kDaorgreatercut-offtoallowmediacomponentsto

branemayhavetobesmaller

mindthat

oducts

withmolecularweightsgreaterthanthecut-offvaluemay

untthatpass

throughdependsonthemembraneporedistributionandthe

the

concentrationprocedure,additionofproteainhibitorsmay

culturesupernatantsshouldbeconcentrated10to20times,

resuspendedinbuffer,andreconcentratedtoremovemedia

oncentrationofsample,proteinispuri-

ssible,affinitychromatography

lumnsandresinsareavailabledepending

onyourneeds(26-31).

Forconcentrationbyprecipitationfromrum-free

media,upolyethyleneglycol(PEG)(32).Ammonium

sulfateprecipitationisnotrecommendedforrecovering

proteinsfromSFM.

PurifyingIntracellularProteins

Toharvestintracellularproducts,cellsarelydmost

respundownat200to

400×gfor10min,let

isresuspendedinalysingbuffer,usuallycontainingsucro

upto0.3M,andproteainhibitorssuchaspepstatinor

phenylmethylsulfonylfluoride(PMSF).

Staudacher(33)employedasimplemethodofsonica-

,onice,

arerepeatedlysonicatedforshortperiods(

˜

10s)after

r

methodforlysingcellswithoutmechanicalforcehasbeen

describedbyEmery(34).

Ifcellsarelydwithdetergent,removedetergentafter

lysistominimizeitsinterferencewithfurtherpurification

elllysis,samplesareusuallyconcentrated

beforefurtherpurification.

18

19

nces

s,erson,D.(1972).9,710.

ws,.(1982)Interviology17,1.

rth,J.L.(1983)ACriticalAppraisalofVirusTaxonomy,Matthews,R.E.F.(ed.),CRCPress,BocaRaton,FL,p.

123.

,G.E.,Summers,r,M.J.(1983)larandCellularBiology3,2156.

,G.E.,Summers,M.D.,andFrar,M.J.(1983).3,2156.

o,C.M.(1975)BaculovirusforInctPestControl:SafetyConsiderations,Summers,M.,Engler,R.,Falcon,L.A.

andVail,P.V.(eds.),AmericanSocietyforMicrobiology,Washington,DC,p.52.

,P.A.,Ayres,e,R.D.(1990)NucleicAcidsRes.18,5667.

,don,M.C.(1992)s38,61.

,e,R.D.(1993)Biotechniques14,810.

,V.A.,Lee,S.C.,Barry,G.F.,andOlins,P.O.(1993).67,4566.

on,D.,Harris,R.,Polayes,D.,Ciccarone,V.,Donahue,R.,Gerard,G.,andJese,J.(1996)Focus17,53.

s,D.,Harris,R.,Anderson,D.,andCiccarone,V.,(1996)Focus18,10.

,J.L.,Goodwin,R.H.,Tompkins,G.J.,andMcCawley,P.(1977)InVitro13,213.

,W.F.,Thomn,D.R.,Davidson,D.J.,Meyer,tellino,F.J.(1991).7,9.

ashi,J.(1982)AdvancesinCellCulture2,133.

,anson,D.R.(1985)Sci.,Vol.C111,ElvierScientificPublishersIrelandLtd.,County

Clare,Ireland,p.19.

,D.,Shauger,A.,andMaiorella,B.(1989)s12,13.

,n,J.(1971).138,432.

,R.J.(1988)AnalyticalBiochem.171,225.

ok,J.,Fritsch,E.F.,andManiatis,T.(1989)MolecularCloning:ALaboratoryManual,ColdSpringHarbor

LaboratoryPress,ColdSpringHarbor.

,S.A.,Whitford,win,G.(1991)ProceedingsoftheEighthInternationalConferenceonInvertebrate

andFishTissueCulture,Frar,M.J.(ed.),TissueCultureAssociation,Columbia,Maryland,p.153.

,K.,Nakajima,Y.,andNatori,S.(1990)Biochem.J.272,633.

,T.,Tessier,D.C.,Richardson,C.,Laliberte,F.,Khouri,H.E.,Bell,A.W.,Storer,A.C.,andThomas,D.Y.(1990)J.

.265,16661.

,K.,Nakajima,Y.,andNatori,S.(1990)Biochem.J.272,633.

,J.B.,White,E.M.,andSito,A.F.(1992)BioPharm5,22.

cher,E.,Kubelka,z,L.(1992)EuropeanJournalofBiochemistry207,987.

na,son,G.S.(1992)LC•GC10,223.

,dy,D.(1992)LC•GC10,356.

,M.W.,Brideau,R.J.,andThomn,D.R.(1989)iousDis.159,255.

,P.,Walker,M.,Wheeler,T.,Bui,P.,Lei,S.,andWeickmann,J.(1992)BioPharm5,28.

,J.,Hancock,W.S.,andWu,S.(1992)LC•GC10,668.

,K.C.(1990).182,301.

,hop,D.H.L.(1987)ProteinEngineeringI,359.

,nett,T.(1992)BioTechniques12,645.

.

BAC-TO-BACProducts:

PlasmidpFASTBAC1ExpressionVector10µg10360-014

pFASTBACDNA

pFASTBAC-gusDNA(4ng)

Manual

pFASTBACHTExpressionVectors10µgeach10584-027

pFASTBACHTa

pFASTBACHTb

pFASTBACHTc

pFASTBACHT-CAT(15ng)

Ni-NTAresin(10ml)

Disposablecolumn(1)

Manual

pFASTBACDUALExpressionVector10µg10712-024

pFASTBACDUALDNA

pFASTBACDUALControlDNA(4ng)

Manual

MAXEFFICIENCYDH10BACCompetentCells5x0.1ml10361-012

CELLFECTINReagent1ml10362-010

BAC-TO-BACExpressionSystems:

BAC-TO-BACBaculovirusExpressionSystem5reactions10359-016

pFASTBAC1ExpressionVector(1each)

MAXEFFICIENCYDH10BACCompetentCells(5x0.1ml)

CELLFECTINReagent(1ml)

BAC-TO-BACHTBaculovirusExpressionSystem5reactions10608-016

pFASTBACHTExpressionVectors(1each)

MAXEFFICIENCYDH10BACCompetentCells(5x0.1ml)

CELLFECTINReagent(1ml)

MolecularGeneticsMediaandAntibiotics:

AmpicillinSodiumSalt,lyophilized5ml13075-015

GentamicinReagentSolution(10mg/ml),liquid10ml15710-015

KanamycinSulfate(100X),liquid100ml15160-013

LBAgar,powder500g22700-025

LBBroth(1X),liquid500ml10855-013

LuriaAgar,powder100g12945-028

10×10ml15544-018

TerrificBroth100g22711-014

SELECTPeptone140500g30392-021

SELECTYeastExtract500g30393-029

SELECTAgar500g30391-023

MolecularBiologyProducts:

1KbDNALadder250µg15615-016

λDNA/HindIIIFragments500µg15612-013

SUBCLONINGEFFICIENCYDH5αCompetentCells2ml18265-017

TaqDNAPolymera,Recombinant100units10342-053

ConcertHighPurityMiniprepSystem25reactions11449-014

100reactions11449-022

ConcertHighPurityMidiprepSystem25reactions11451-010

50reactions11451-028

ConcertHighPurityMaxiprepSystem10reactions11452-018

25reactions11452-026

20

dProducts

21

.

Reagents:

4%AgaroGel40ml18300-012

Agaro100g15510-019

Bluo-gal100mg15519-010

Buffer-SaturatedPhenol100ml15513-039

EthidiumBromide1g15582-018

EthylenediaminetetraaceticAcid100g15576-010

IPTG1g15529-019

MUG100mg10215-010

Phosphate-BufferedSaline(PBS),pH7.4(1X)500ml10010-015

Phosphate-BufferedSaline(PBS),pH7.4(10X)500ml70011-036

10%SDSSolution4×100ml15553-019

10XTAEBuffer1L15558-042

1MTris-HCl,pH8.01L15568-026

X-Glucuronide100mg10214-013

NucleicAcidPurificationRackeach11494-010

InctMedia:

FetalBovineSerum,qualified,heat-inactivated100ml16140-014

Grace’sInctMedium(1X),liquid500ml11595-022

IPL-41InctMedium(1X),liquid500ml11405-024

NeutralRedSolution100ml15330-012

Penicillin-Streptomycin,liquid100ml15070-014

PLURONIC®F-68,10%(100X)100ml24040-016

Sf-900IISFM(1X),liquidwithL-glutamine500ml10902-013

Sf-900IISFM(1X),methionineandcystine-free500ml21012-018

Sf-900IISFM(1.3X),liquidwithL-glutamine100ml10967-016

TC-100InctMedium,powder10×1L11600-061

22

ationsDataforInctCellLinesGrowninSerum-FreeMedium

MonolayerculturesofSf9,Sf21,andTn-368cellsin

Grace’ssupplementedmediumplus10%heat-

inactivatedFBSwereadaptedtosuspension

cultureasdescribedinProtocol2,andthentorum-free

growthinSf-900IISFMusingthedirectadaptationmethod

olayerBTI-TN-5B1-4

culturewasadaptedtogrowthinSf-900IISFM,thento

suspensioncultureinthesamemedium,andfinallyto

EXPRESS-FIVESFM.

Followingaminimumof10concutivepassagesin

eachmedium,thefourcelllineswereededin35-to

150-mlshakeflasksorspinnerculturesat2×105to3×105

viablecells/eswereincubatedat27°Cwith

stirringspeedsof90to100rpmforspinnerflasksand

sintable7repre-

ntmaximumcelldensitiesinsmall-scalesuspension

culturesondays4to7post-planting.

mcelldensitiesin

small-scalesuspensionculture.

GrowthMedium:

CellGrace’sTNM-FH+10%FBSSf-900IISFMEXPRESS-FIVESFM

line(viablecells/ml×10

6

)(viablecells/ml×10

6

)(viablecells/ml×10

6

)

Sf94to68to12—

Sf213to55to7—

Tn-3682to33to5—

BTI-TN-5B1-4—3to44to5

Comments

•Tn-368cellsusuallymaintaintheircharacteristic

spindlemorphologyundersuspensionconditionsifthe

growthmediumismaintainedwithinoptimalpHand

osmolalityranges.

•UnliketheSf9orSf21celllines,Tn-368andBTI-TN-

5B1-4culturesoftendierapidlyuponreaching

maximumcelldensityandaredifficulttorecoverif

viabilitiesdropbelow50%.Toavoidproblems,cultures

ofTn-368andBTI-TN-5B1-4cellsshouldbesplit

frequentlywhileinmid-exponentialgrowth.

ExpressionofRecombinantProteininSmall-

ScaleCulture

Shakeflaskcultures(50-to100-ml)ofSf9,Tn-368,and

BTI-TN-5B1-4cellswereadaptedtogrowthinrum-freeor

tureswereinfected

withrAcNPV(CloneVL-941)expressingrecombinantb-

galactosidaatthefollowingdensitiesandMOIs:

Sf9cells:2.5×106viablecells/mlMOI=5.0

Tn-368cells:1.0×106viablecells/mlMOI=5.0

BTI-TN-5B1-4

cells:1.5×106viablecells/mlMOI=4.0

Cultureswereincubatedpost-infectionat27°Cwitha

inantβ-galactosida

activitywasmonitoredthroughday4or5post-infectionfor

sareshownintable8.

Comments

•Recommendedinfectiondensitiesarelowerforthe

Tn-368andBTI-TN-5B1-4celllinesbecauinrum-

freesuspensionculturethecellsgenerallyattainlower

maximumdensities(5×106to6×106viablecells/ml)

thanSf9cells(8×106to12×106viablecells/ml).Infect

cultureswhileinmid-exponentialgrowth(population

doublingtimesof16to24h)atcelldensitiesnogreater

than40%ofthemaximumnormallyobrvedforoptimal

expression.

•Recombinantproteinexpressionvariesfordifferent

proteins,andtheoptimalcellsforeachproteincanvary.

GrowthandExpressionofRecombinant

ProteinsinLarge-ScaleCulture

ForscaleupofarecombinantproductusingBEVS

technology,itisimportanttodeterminewhetherthemedium

(rum-supplementedorrum-free)willadequately

supportscale-up,aswellasdownstreamprocessing

considerations(i.e.,cellparationandproductpurifica-

tion).

Thedataintable9compareresultsofpilot-scalecell

growthandexpressionofrecombinantproteinsinSf-900II

inant

productyieldsreachedorexceededlevelsobtainedunder

tyieldswereupto10-fold

higherwithSf-900IISFMthanthoproducedunder

rum-supplementedconditionsanddisplayacceptable

glycosylationorbioactivity.

ComparisonofrAcNPVTiterinSmall-Scale

SuspensionCulture

Shakeflaskcultures(75-ml)ofSf9andBTI-TN-5B1-4

tures

wereinfectedwithrAcNPV(CloneVL-941)expres-sing

recombinantβ-cateculturesforeach

wereinfectedat1×106viablecells/mlatanMOIof0.10.

Cultureswereinfectedat27°Cwithastirringspeedof

turesweresampledat24,48,and72h

iedsupernatantsamplesweretiteredby

sareshownintable10.

Comments

•ForBTI-TN-5B1-4cultures,maximumrAcNPVtiters

tunusual

forBTI-TN-5B1-4cellstoproducevirusstocks1to

counteractthis,maintainandproduceyourworking

rAcNPVstocksinSf9orSf21cellsanduthe

BTI-TN-5B1-4celllineforexpressionofrecombinant

products.

23

titersinsmall-scalesuspensionculture.

Virustiterpost-infection(pfu/ml)

CelllineMedium24h48h72h

Sf9Grace’sTNM-FHsupplementedwith1×1085×1086×108

10%FBS

Sf9Sf-900IISFM5×1073×1084×108

BTI-TN-5B4-1EXPRESS-FIVESFM2×1056×1065×106

-scalerecombinantproteinexpressionincellscultured.

Expressionlevel

RecombinantproteinBioreactorInSf-900IISFMInrumcontrol

α-Galactosida2-LCelligen4,700U/ml2,500–5,000U/ml

30-LChemapairlift5,040U/ml

β-Galactosida5-LCelligen240,000U/ml150,000U/ml

Erythropoietin2-LCelligen7,800U/ml1,000–2,000U/ml

5-LCelligen6,500U/ml

HantaanSnucleocapsid5-LCelligen5-foldhigherthan

rumcontrol*

Humanchoriogonadotropin5-LCelligen8,192–8,345ng/ml768–1,075ng/ml

inmonolayer

Leukemiainhibitoryfactor10-LBraun9µg/

rVP6,rotaviruscapsidprotein5-LCelligen118µg/ml20µg/mlin

IPL-41with10%FBS

*Specificproductyieldnotprovided.

TABLE8.

β

-galactosidaexpressioninsmall-scalesuspensionculture.

Sf9cellsTn-368BTI-TN-5B1-4cells

Grace’sTNM-FHGrace’sTNM-FHSf-900IISf-900IIEXPRESS-FIVE

Dayspost-infection+10%FBSSf-900IISFM+10%FBSSFMSFMSFM

1––––6899

2––––––––1628

395254256179252

4276550––––499798

51985832669––––

Note:Dataareunitsβ-gal/ml×103.

24

Notes:

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