Proteins,thebuildingblocksoflife,aresynthesizedbyalllivingformsaspart oftheirnaturalmetabolism.Someproteins,suchasenzymes,serveasbiocatal ystsandincreasetherateofmetabolicreactions,while
Trang 1ContentslistsavailableatScienceDirect BiotechnologyAdvances jou r n a lh o m e pag e :www.e l s ev i e r.co m/lo c a t e /b io te c h ad v
Researchreviewpaper
Productionofrecombinantproteinsbymicrobesandhigherorganisms
ArnoldL.Demaina,⁎ ,PreetiVaishnavb
a Resea
rchInstituteforScientistsEmeriti(R.I.S.E.),DrewUniversity,Madison,NJ07940,USA
b 206AkshardeepApts.,NearNewJainTemple,GIDC,Ankleshwar393002,Gujarat,India
a r t i c l ein f o
Articlehistory:
Received26September2008
Receivedinrevisedform14January2009
Accepted21January2009
Availableonline31January2009
Keywords:recombinan
tproteinsenzymes
bacteria
yeasts
filamentousfungiin
sectcellsmammalia
ncellstransgenicani
mals
transgenicplants
a b s t r a c t Largeproteinsareusuallyexpressedinaeukaryoticsystemwhilesmalleronesareexpressedinprokaryoticsystems.Fo rproteinsthatrequireglycosylation,mammaliancells,fungiorthebaculovirussystemischosen.Theleastexpensive,e asiestandquickestexpressionofproteinscanbecarriedoutinEscherichiacoli.However,thisbacteriumcannotexpress
verylargeproteins.Also,forS–Srichproteins,andproteinsthatrequirepost-translationalmodifications,E.coliisnotthesystemofchoice.ThetwomostutilizedyeastsareSaccharomycescerevisia eandPichiapastoris.Yeastscanproducehighyieldsofproteinsatlowcost,proteinslargerthan50kDcanbeproduced,signa lsequencescanberemoved,andglycosylationcanbecarriedout.Thebaculoviralsystemcancarryoutmorecomplexpos
t-translationalmodificationsofproteins.Themostpopularsystemforproducingrecombinantmammalianglycosylat edproteinsisthatofmammaliancells.Geneticallymodifiedanimalssecreterecombinantproteinsintheirmilk,bloodo rurine.Similarly,transgenicplantssuchasArabidopsisthalianaandotherscangeneratemanyrecombinantproteins.
©2009ElsevierInc.Allrightsreserved.
Contents
1.Introduction 297
2.Enzymeproduction 298
3.Systemsforproducingrecombinantproteins 298
3.1.Bacteria 299
3.1.1.E.coli 299
3.1.2.Bacillus 300
3.1.3.Otherbacteria 300
3.2.Yeasts 300
3.3.Filamentousfungi(molds) 302
3.4.Insectcells 302
3.5.Mammaliancells 302
3.6.Transgenicanimals 303
3.7.Transgenicplants 304
4.Conclusions 304
References 305
1.Introduction
⁎Correspondingauthor.DrewUniversity,R.I.S.E.,HS-330,Madison,NJ007940,USA.Tel.:
+19734083937;fax:+19734083504.
E-mailaddress: ademain@drew.edu (A.L.Demain).
Proteins,thebuildingblocksoflife,aresynthesizedbyalllivingformsaspart oftheirnaturalmetabolism.Someproteins,suchasenzymes,serveasbiocatal ystsandincreasetherateofmetabolicreactions,whileothersformthecytosk eleton.Proteinsplayasignificantroleincellsignaling,immuneresponses,cel ladhesion,
0734-9750/$–
seefrontmatter©2009ElsevierInc.Allrightsreserved.doi: 10.1016/
j.biotechadv.2009.01.008
Trang 229 29
andthecellcycle.Theyarecommerciallyproducedinindustrieswiththeaidofg
eneticengineeringandproteinengineering.Nativeandrecombinantprotei
nsbenefitmajorsectorsofthebiopharmaceuticalindustry,theenzymeindu
stry,andtheagriculturalindustry.Productsoftheseindustriesinturnaugme
ntthefieldsofmedicine,diagnostics,food,nutrition,detergents,textiles,leat
her,paper,pulp,polymersandplastics.Thefirstproteinvaccineproducedwa
sthecow-poxvaccinebyJennerin1796.Themicrobialfermentationindustrywasborni
ntheearly1900swhenthefirstlarge-scaleanaerobicfermentationstomanufacturechemicalssuchasacetoneand
butanolbegan,followedbytheaerobicproductionofcitricacid.Penicillinwas
discoveredin
1927butitsdevelopmentdidnotoccuruntilthestartofthe1940s,priortothetimet
hatstreptomycinwasdiscovered.Thefirstproteinpharmaceuticalproduce
dwasinsulinbyBantingandBestin1922.Themodernbiotechnologyerabegan
in1971withtheestablishmentoftheCetusCorporationinCaliforniaabout1–
2yearsbeforethediscoveryofrecombinantDNAbyBerg,CohenandBoyerinC
alifornia.Thiswasfollowed5yearslaterbythestartofGenentech,andthenbyot
hercorporationssuchasAmgenandBiogen,etc
By2002,over155approvedpharmaceuticalsandvaccineshadbeendeve
lopedbybiopharmaceuticalcompanies.Today,morethan
200approvedpeptideandproteinpharmaceuticalsareontheFDAlist.Someof
therecombinantproteinpharmaceuticalsproducedarehumaninsulin,al
bumin,humangrowthhormone(HGH),FactorVIII,andmanymore.Biophar
maceuticalshavebeeninstrumentalinradi-callyimprovinghumanhealth(Swartz,1996):
(i)diabeticsnolongerhavetofearproducingantibodiestoanimalinsulin;
(ii)childrendeficientingrowthhormonenolongerhavetosufferfromdwarfis
morfeartheriskofcontractingKreutzfeld–Jacobsyndrome;(iii)chil-drenwhohavechronicgranulomatousdiseasecanleadanormallifebytakingg
ammainterferontherapy;and(iv)patientsundergoingcancerchemothera
pyorradiationtherapycanrecovermorequicklywithfewerinfectionswhe
ntheyusegranulocytecolony-stimulatingfactor(G-CSF).Manyotherexamplesoftheconquestofdiseasecouldbementioned
2.Enzymeproduction
Theenzymeindustryflourishedinthe1980sand1990swhenmicrobiale
nzymescameontothescene.Inthe1970s,mostoftheenzymesusedweretradit
ionallyderivedfromplantandanimalsources,whichresultedinalowlevelofa
vailability,highprices,andstuntedgrowthoftheenzymeindustry.Microbiale
nzymesprovedeconomicallyfavorablesincecultivationofmicrobeswasmu
chsimplerandfasterthanthatofplantsandanimalsandtheproducingorgani
smscouldbeeasilymanipulatedgeneticallytoproducedesiredqualitiesand
quantitiesofenzymes.Someofthemajorindustrialusesofenzymesinmanuf
acturinginclude(1)Escherichiacoliamidasetoproduce6-aminopenicillanicacid(6-APA)at40,000tons/year;
(2)StreptomycesxyloseisomerasetoisomerizeD-glucosetoD
-fructoseat100,000tons/
year;and(3)Pseudomonaschlorapisnitrilehydratasetoproduceacrylamide
fromacrylonitrileat30,000tons/
year(Jaegeretal.,2002).Amylasesareproducedatanannualrateof95,000ton
speryear.Thetotalmarketforindustrialenzymesreached$2billionin2000an
dhasrisento$2.5billiontoday.Theleadingenzymeisproteasewhichaccount
sfor57%ofthemarket.Othersincludeamylase,glucoamylase,xyloseisomer
ase,lactase,lipase,cellulase,pullulanaseandxylanase.Thefoodandfeedindu
striesarethelargestcustomersforindustrialenzymes.Overhalfoftheindustri
alenzymesaremadebyyeastsandmolds,withbacteriaproducingabout30
%.Animalsprovide8%andplants4%.Enzymesalsoplayakeyroleincatalyzingr
eactionswhichleadtothemicrobialformationofantibioticsandothersecond
arymetabolites
Overtheyears,highertitersofenzymeswereobtainedusing“brute
force”mutagenesisandrandomscreeningofmicroorganisms.Recom-binantDNAtechnologyactedasaboonfortheenzymeindustryinthe
followingways(Falch,1991):
(i)plantandanimalenzymescouldbemadebymicrobialfermentations,e.g.,c hymosin;
(ii)enzymesfromorganismsdifficulttogroworhandlegeneticallywerenow producedbyindustrialorganismssuchasspeciesofAspergillusandTrichoder ma,andKluyveromyceslactis,Saccharomycescerevisiae,Yarrowialipolyticaan dBacilluslicheniformis(e.g.,thermophiliclipasewasproducedbyAspergilluso
ryzaeandThermoanaerobactercyclodextringlycosyltrans-ferasebyBacillus);
(iii)enzymeproductivitywasincreasedbytheuseofmultiplegenecopies,stro ngpromotersandefficientsignalsequences;
(iv)productionofausefulenzymefromapathogenicortoxin-producingspeciescouldnowbedoneinasafehost;and(v)proteinengineering wasemployedtoimprovethestability,activityand/
orspecificityofanenzyme
Bythe1990s,manyenzymeswereproducedbyrecombinanttechniques In1993,over50%oftheindustrialenzymemarketwasprovidedbyrecombina ntprocesses(Hodgson,1994);saleswere
$140million(Stroh,1994 ).Plantphytase,producedinrecombinantAs- pergillusnigerwasusedasafeedfor50%ofallpigsinHolland.A1000-foldincreaseinphytaseproductionwasachievedinA.nigerbytheuseofrecom binanttechnology(VanHartingsveldtetal.,1993).Industriallipaseswerecl onedinHumicolaandindustriallyproducedbyA.oryzae.Theyareusedforlaund
rycleaning,inter-esterificationoflipidsandesterificationofglucosides,producingglycolipid
swhichhaveapplica-tionsasbiodegradablenon-ionicsurfactantsfordetergents,skincareproducts,contactlensesandasfood emulsifiers.MammalianchymosinwasclonedandproducedbyA.nigerorE.c oliandrecombinantchymosinwasapprovedintheUSA;itspricewashalfthato fnaturalcalfchymosin.Over60%oftheenzymesusedinthedetergent,foodan dstarchprocessingindustrieswererecombinantproductsasfarbackasthe mid-1990s(Cowan,1996)
Today,withtheaidofrecombinantDNAtechnologyandproteinengineeri
ng,enzymescanbetailor-madetosuittherequirementsoftheusersoroftheprocess.Itisnolongernecessa rytosettleforanenzyme'snaturalproperties.Enzymesofsuperiorqualityha
vebeenobtainedbyproteinengineering,specificallybysite- directedmuta-genesis.SinglechangesinaminoacidsequencesyieldedchangesinpHoptim
um,thermostability,feedbackinhibition,carbonsourceinhibi-tion,substratespecificity,Vmax,KmandKi.Anewandimportantmethodforim provingenzymeswasdirectedevolution(alsoknownasappliedmoleculare volutionordirectedmolecularevolution)(
Kuch-nerandArnold,1997;Arnold,1998;JohannesandZhao,2006).Unlikesitedirect edmutagenesis,thismethodofpoolingandrecombiningpartsofsimilargen esfromdifferentspeciesorstrainsyieldsremarkableimprovementsinenzy mesinaveryshortamountoftime.Theprocedureactuallymimicsnatureintha tmutation,selectionandrecombinationareusedtoevolvehighlyadaptedprot eins,butitismuchfasterthannature.Thetechniquecanbeusedtoimprovepro teinpharmaceuticals,smallmoleculepharmaceuticals,genetherapy,DNAv accines,recombinantproteinvaccines,viralvaccinesandtoevolveviruses.P roteinsfromdirectedevolutionworkwerealreadyonthemarketin2000(Tobi netal.,2000)
Manyenzymesareusedastherapeuticagentstotreatgastro- intestinalandrheumaticdiseases,thromboses,cysticfibrosis,meta-bolicdiseaseandcancer.Salesoftherapeuticenzymeswere
$2.3billionin1996whilein1998marketsfortherapeuticenzymeswereasfollo ws(Stroh,1999):Pulmozyme(DNase)forcysticfibrosis,acutemyocardialinf arctionandischemicstroke,$350million;Ceredase®and
Cerezyme®(
r-DNAversion)forGaucher'sdisease,
$387million.By2007,themarketforCerezyme®reached$1.1billion.Thether apeuticmarketisinadditiontotheindustrialenzymemarketdiscussedabo ve
3.Systemsforproducingrecombinantproteins Bymeansofgeneticengineering,desiredproteinsaremassivelygenerat edtomeetthecopiousdemandsofindustry.Hence,most
Trang 3binantproteinproductionisgettingthedesiredDNAcloned;thentheprotei
nisamplifiedinthechosenexpressionsystem.Thereisawidevarietyofprotein
expressionsystemsavailable.Proteinscanbeexpressedincellculturesofba
cteria,yeasts,molds,mammals,plantsorinsects,orviatransgenicplantsan
danimals.Proteinquality,functionality,productionspeedandyieldarethem
ostimportantfactorstoconsiderwhenchoosingtherightexpressionsyste
mforrecombinantproteinproduction
Asof2002,therewereabout140therapeuticproteinsapprovedinEurop
eandtheUSA(Walsh,2003
).Non-glycosylatedproteinsareusuallymadeinE.colioryeastsandtheyconstitute
40%ofthetherapeuticproteinmarket.N-glycosylatedproteinsareusuallymadeinmammaliancellswhichmimichum
anglycosylation.Chinesehamsterovary(CHO)cellsprovideabout50%ofthe
therapeuticproteinmarketbuttheprocessisveryexpensiveandtheglycopr
oteinsmadearenotexactlythehumantype,andinsomecases,theymustbem
odified.Yeasts,moldsandinsectcellsaregenerallyunabletoprovidemamm
alianglycosylation.However,thepopularmethylotrophicyeast,Pichiapast
oris,hasbeengeneticallyengineeredtoproduceahumantypeofglycosylatio
n(seebelow)
3.1.Bacteria
3.1.1.E.coli
E.coliisoneoftheearliestandmostwidelyusedhostsfortheproductionofh
eterologousproteins(Terpe,2006).Advantagesanddisadvantagesareshow
ninTable1.Theseincluderapidgrowth,rapidexpression,easeofcultureandhi
ghproductyields(Swartz,1996).Itisusedformassiveproductionofmanycom
mercializedproteins.Thissystemisexcellentforfunctionalexpressionofno
n-glycosylatedproteins.E.coligeneticsarefarbetterunderstoodthanthoseof
anyothermicroorganism.Recentprogressinthefundamentalunder-standingoftranscription,translation,andproteinfoldinginE.coli,together
withtheavailabilityofimprovedgenetictools,ismakingthisbacteriummor
evaluablethaneverfortheexpressionofcomplexeukaryoticproteins.Itsgen
omecanbequicklyandpreciselymodifiedwithease,promotorcontrolisnotdi
fficult,andplasmidcopynumbercanbereadilyaltered.Thissystemalsofeatu
resalterationofmetaboliccarbonflow,avoidanceofincorporationofamino
acidanalogs,formationofintracellulardisulfidebonds,andreproduciblepe
rfor-mancewithcomputercontrol.E.colicanaccumulaterecombinantproteins
upto80%ofitsdryweightandsurvivesavarietyofenvironmentalconditions
TheE.colisystemhassomedrawbacks,however,whichhavetobeoverco
meforefficientexpressionofproteins.Highcelldensitiesresultintoxicitydue
toacetateformation;however,thiscanbeavoidedbycontrollingthelevelofox
ygen.Proteinswhichareproducedasinclusionbodiesareofteninactive,inso
lubleandrequirerefolding.Inaddition,thereisaproblemproducingprotein
swithmanydisulfidebondsandrefoldingtheseproteinsisextremelydifficu
lt.TheE.colisystemproducesunmodifiedproteinswithoutglycosylationwh
ichisthereasonwhysomeproducedantibodiesfailtorecognizemamma-lianproteins(JenkinsandCurling,1994
).Surprisingly,thenon-glycosylatedhumantPAproducedinE.coliwasfullyactiveinvitro
Table1
CharacteristicsofE.coliexpressionsystems
AdvantagesDisadvantages
RapidexpressionProteinswithdisulfidebondsdifficulttoexpress
HighyieldsProduceunglycosylatedproteins
(Sarmientosetal.,1989 ).DespitethelackoftheusualtPAglycosyla-
tion,theproducthadafour-foldlongerhalf-lifeinplasmaandacorrespondinglongerclearancerateinanimals(Dartaretal ,1993).Theamountproducedwas5–10%oftotalE.coliprotein
ToimprovetheE.coliprocesssituation,thefollowingmeasureshavebeen taken:(i)useofdifferentpromoterstoregulateexpression;
(ii)useofdifferenthoststrains;(iii)co-expressionofchaperonesand/ orfoldases;(iv)loweringoftemperature;
(v)secretionofproteinsintotheperiplasmicspaceorintothemedium; (vi)reducingtherateofproteinsynthesis;(vii)changingthegrowthmedium; (viii)additionofafusionpartner;
(ix)expressionofafragmentoftheprotein;and(x)invitrodenaturationandref oldingoftheprotein(Swartz,2001;ChoiandLee,2004;Mergulhaoetal.,2005
;ShiloachandFass,2005;Maldonadoetal.,2007;Chou,2007;Wongetal.,200 8
HighcelldensityfermentationsofE.colihaveresultedindrycellcontentso f20to175g/
l(Lee,1996).Theacetateproductionandtoxicityproblemcanbesolvedbyfeed ingglucoseexponentially,andkeepingthespecificgrowthratebelowthatwh ichbringsonacetateproduction.Inthisway,yieldsashighas5.5g/Lofα-consensusinterferoninbrothwereattained(Fieshko,1989).Growthinalong
-termchemostat(219generationsunderthelowdilutionrateof0.05h−1)yiel dedanE.colimutantthathadanincreasedspecificgrowthrate,increasedbio massyields,shorterlagphase,lessacetateproductionandincreasedresistan cetostress(Weikertetal.,1997).Thisstrainproducedincreasedlevelsofsecre tedheterologousproteins(Weikertetal.,1998)
HeterologousproteinsproducedasinclusionbodiesinE.coliareinactive ,aggregatedandinsoluble,usuallypossessingnon-nativeintra-andinter-moleculardisulfidebondsandunusualfreecysteines(Fischeretal.,1993).To obtainactiveprotein,thesebodiesmustberemovedfromthecell,theproteins solubilizedbydenaturantswhichunfoldtheproteins,anddisulfidebondsm ustbeeliminatedusingreducingagents.Refoldingisaccomplishedbytherem ovalofthedenaturantandthereducingagent,followedbyrenaturationofthe protein.Renaturationprocessesusedinclude(i)airoxidation,
(ii)theglutathionereoxidationsystem,and(iii)themixeddisulfidesofprotei
n-S-sulfonateandprotein-S-glutathionesystem.Heterologousrecombinantproteinscanbemadeinbiol ogicallyactivesolubleformathighlevelswhentheirgenesarefusedtotheE.col ithioredoxingene(LaVallieetal.,1993 ).MurineIL-2,humanIL-3,murineIL- 4,murineIL-5,humanIL-6,humanM1P-lalpha,humanIL-11,humanM-CSL,murineL1F,murineSFandhumanBMP-2areproducedatlevelsof5– 20%oftotalproteinsasfusionsinE.colicytoplasm.Somefusionsretainthethi oredoxinpropertiesofbeingreleasedbyosmoticshockorfreeze/
thawmethods,andhighthermalstability.Thioredoxinissmall(11kD)andisno rmallyproducedat40%oftotalcellproteininsolubleform(Lunnetal.,1984).An otherusefulmethodofreducingtheformationofinclusionbodiescontainin gheterologousproteinsistolowerthetemperatureofgrowthfrom 37°Cto30°C(Schein,1989)
Higheryieldsarenormallyproducedinthecytoplasmthanintheperiplas micspace.Cytoplasmicproteinscanbeexportedtosimplifypurificationandf acilitatecorrectfolding.Thismustbedonewithproteinscontainingdisulfide bondssincethecytoplasmistooreducinganenvironment.Tosecretethese proteinsintotheperiplasm,afusionismadewithaleaderpeptideattheN-terminus.Togettheproteinsoutoftheperiplasmandintothemedium,osmotic shockorcellwallpermeabilizationisused.Toincreaseproduction,apromoter system(lac,tac,trc)isused.Promotersystemsmustbestrongandtightlyregul
atedsothattheyhavealow-basallevelofexpression,easilytransferabletootherE.colistrains,andhaveasi mpleandinexpensiveinductiontechnique,independentofmediaingredie nts
Easeofcultureandgenomemo
difications Proteinsproducedwithendotoxins
SecretionofrecombinantproteinsbyE.coliintotheperiplasmorinto themediumhasmanyadvantagesoverintracellularproductionas
InexpensiveAcetateformationresultingincelltoxicity inclusionbodies.Ithelpsdownstreamprocessing,foldingandinvivo
Massproductionfastandcosteffe
ctive
Proteinsproducedasinclusionbodies,areinactive;
requirerefolding. stability,andallowstheproductionofsoluble,activeproteinsatareducedpr
ocessingcost(Mergulhaoetal.,2005).Highlevelexcretion
Trang 430 30
Table2
AdvantagesofBacillusexpressionsystems
Strongsecretionwithnoinvolvementofintracellularinclusionbodies
Easeofmanipulation
Geneticallywellcharacterizedsystems
Highlydevelopedtransformationandgenereplacementtechnologies.Supe
riorgrowthcharacteristics
Metabolicallyrobust
Generallyrecognizedassafe(GRASstatus)byUSFDAEfficie
ntandcosteffectiverecovery
hasbeenobtainedwiththefollowingheterologousproteins:PhoA(alkalin
ephosphatase)at5.2g/
Lintotheperiplasm;LFT(levanfructotransferase)at4g/
Lintothemedium;hGCSF(humangranulocytecolony-stimulatoryfactor)at3.2g/
Lintotheperiplasm;cellulosebindingdomainat2.8g/
Lintotheperiplasm;IGF-1(insulin-likegrowthfactor)at2.5g/
Lintotheperiplasm;choleratoxinBat1g/
Lintothemedium(Mergulhaoetal.,2005).Asearlyas1993,recombinantproc
essesinE.coliwereresponsibleforalmost$5billionworthofproducts,i.e.,ins
ulin,humangrowthhormone,α,β,γ-interferonsandG-CSF(Swartz,1996)
3.1.2.Bacillus
OtherusefulbacterialsystemsarethoseoftheGram-positivebacilli.Thesearemainlypreferredforhomologousexpressionofenzy
messuchasproteases(fordetergents)andamylases(forstarchandbaking).So
meadvantagesofusingBacillussystemsareshowninTable2.Someoftheseadv
antagesareonlypresentinindustrialstrainswhichareoftenunavailabletoaca
demicresearchers.Inaddition,thegenomesofBacillussubtilisandB.lichenifo
rmishavebeensequenced,andthereisnoproductionofharmfulexotoxinsore
ndotoxins.Thesecretionofthedesiredproteinsintothefermentationmediu
mresultsineasydownstreamprocessing,eliminatingtheneedforcelldisrupt
ionorchemicalprocessingtechniques.Thismakesrecoveryrelativelyeffici
entandcost-effective.ThespeciesgenerallyusedforexpressionareBacillusmegaterium,
B.subtilis,B.licheniformisandBacillusbrevis.Theydonothavelipopolysacchar
ide-containingoutermembranesasdoGram-negativebacteria.IndustrialstrainsofB.subtilisarehighsecretorsandhoststr
ainsusedforsuccessfulexpressionofrecombinantproteinsareoftendeleted
forgenesamyE,aprE,nprE,spoIIAC,srfCandtransformedvianaturalcompe-tence.Bacillusproteinyieldsareashighas3g/L
ThereisaproblemwithB.subtilisbecauseofitsproductionofmanyprotea
seswhichsometimesdestroytherecombinantproteins.Theyincludeseven
knownproteases(Heetal.,1991),fiveofwhichareextracellular:
(i)Subtilisin(aprEgene):majoralkalineserineprotease
(ii)Neutralprotease(nprE):majormetalloprotease,containsZn
(iii)Minorserineprotease(epr);inhibitedbyphenylmethanesulfo-nylfluoride(PMSF)andethylenediaminetetraaceticacid
(EDTA)
(iv)BacillopeptidaseF(bpf):anotherminorserineprotease/ester-ase;inhibitedbyPMSF
(v)Minormetalloesterase(mpe)
(vi)ISP-I(isp-I):majorintracellularserineprotease,requiresCa
(vii)ISP-II(isp-II):minorintracellularserineprotease
Thefirsttwoenzymesaccountfor96–
98%oftheextracellularproteaseactivity.Otherresearchgroupshavereporte
dsixtoeightextracellularproteases.Wuetal
(1991)removedsixandonly0.32%activityremained.Growthinthepresence
of2mMPMSFeliminatedalltheproteaseactivity.AB.subtilisstrainhasbeend
evelopedforgeneticengineeringwhichisdeficientineightextracellularpro
teases(Murashimaetal.,2002).Carehastobetakenwithregardtoexcessivegr
owthratesandaeration.Productionofextracellularhumanalpha
interferonbyB.subtilisisrepressedbyhighgrowthrateandbyexcessoxygen( MeyerandFiechter,1985)
Anexoprotease-deficientB.licheniformishoststrainhasbeenspecificallytailoredforheterolo
gousgeneexpression.Itisaspor-ogenousandgiveshighextracellularexpressionlevelswithminimallossofpr oductduetoproteolyticcleavagesubsequenttosecretion.Toobtainamorege neticallystablesystemaftertransformationandtoincreaseproductionlevel
s,theα-amylasegenehasalsobeenremoved.Acomparisonofhostorganismswasma
deforproductionofinterleukin-3(vanLeenetal.,1991)amongE.coli,B.licheniformis,S.cerevisiae,K.lactisandC1 27mammaliancells.ThebestsystemwasreportedtobeB.licheniformis B.brevisisalsousedtoexpressheterologousgenesduetoitsmuchlowerpro teaseactivityandproductionofaproteinaseinhibitor(UdakaandYamagata,1
994).HumanepidermalgrowthfactorwasproducedinB.brevisatalevelof3g/ L(Ebisuetal.,1992)
HeterologousproteinssuccessfullyexpressedinBacillussystemsinclud
einterleukin-3EGFandesterasefromPseudomonas.Homolo-gousproteinsincludeBacillusstearothermophilusxylanase,naproxenester ase,amylasesandvariousproteases
3.1.3.Otherbacteria AnimprovedGram-negativehostforrecombinantproteinproduc-tionhasbeendevelopedusingRalstoniaeutropha(Barnardetal.,
2004.)ThesystemappearssuperiortoE.coliwithrespecttoinclusionbodyfor
mation.Organophosphohydrolase,aproteinpronetoinclu-sionbodyformationwithaproductionoflessthan100mg/
LinE.coli,wasproducedat10g/
LinR.eutropha.ThePfenexsystemusingPseudomonasfluorescenshasyielded
4g/LoftrimericTNF-alpha(SquiresandLucy,2008).Staphylococcuscarnosuscanproduce2g/ LofsecretedmammalianproteinwhereasthelevelmadebyStreptomyceslivi dansis0.2g/L(Hanssonetal.,2002)
3.2.Yeasts Yeasts,thesingle-celledeukaryoticfungalorganisms,areoftenusedtoproducerecombinantpr oteinsthatarenotproducedwellinE.colibecauseofproblemsdealingwithfo ldingortheneedforglycosylation.Themajoradvantagesofyeastexpressions ystemsarelistedinTable3.Theyeaststrainsaregeneticallywellcharacterized andareknowntoperformmanyposttranslationalmodifications.Theyareeas ierandlessexpensivetoworkwiththaninsectormammaliancells,andareeasily adaptedtofermentationprocesses.ThetwomostutilizedyeaststrainsareS.ce revisiaeandthemethylotrophicyeastP.pastoris.Variousyeastspecieshavepro ventobeextremelyusefulforexpressionandanalysisofrecombinanteukary oticproteins.Forexample,A.nigerglucoseoxidasecanbeproducedbyS.cerev isiaeat
9g/L
S.cerevisiaeofferscertainadvantagesoverbacteriaasacloninghost(Gellis onetal.,1992).(i)Ithasalonghistoryofuseinindustrialfermentation
(ii)Itcansecreteheterologousproteinsintothe
Table3 Advantagesofyeastexpressionsystems Highyield
Stableproductionstrains Durability
Costeffective Highdensitygrowth Highproductivity Suitabilityforproductionofisotopically-labeledprotein RapidgrowthinchemicallydefinedmediaProductp rocessingsimilartomammaliancellsCanhandleS–
Srichproteins Canassistproteinfolding
Trang 5ructuralgenes
(iii)Itcarriesoutglycosylationofproteins.However,glycosylationbyS.cerev
isiaeisoftenunacceptableformammalianproteinsbecausetheO-linkedoligosaccharidescontainonlymannosewhereashighereukaryotic
proteinshavesialylatedO-linkedchains.Furthermore,theyeastover-
glycosylatesN-
linkedsitesleadingtoreductioninbothactivityandreceptor-binding,andmaycauseimmunologicalproblems.Productsonthemarketwh
icharemadeinS.cerevisiaeareinsulin,hepatitisBsurfaceantigen,urateoxid
ase,glucagons,granulocytemacrophagecolonystimulatingfactor(GM-CSF),hirudin,andplatelet-derivedgrowthfactor
Almostallexcretedeukaryoticpolypeptidesareglycosylated.Glycosyl
ationisspecies-,tissue-andcell-type-specific(Parekh,1989).Insomecases,anormallyglycosylatedproteinisactiv
ewithoutthecarbohydratemoietyandcanbemadeinbacteria.Thisisthecase
withγ-interferon(Rinderknechtetal.,1984).Incaseswhereglycosylationisnecess
aryforstabilityorproperfolding(e.g.,erythropoietinandhumanchorionicgo
nadotropin),thiscanoftenbeprovidedbyrecombinantyeast,mold,insector
mammaliancells.MammaliansecretedproteinsareglycosylatedwithD
-mannosesugarscovalentlyboundtoaspar-agine-linkedN-acetyl-D
-glucosaminemolecules.Fungalenzymeswhichareexcretedoftenshowthes
ametypeofglycosylation(ElbeinandMolyneux,1985),althoughadditional
carbohydrateslinkedtotheoxygenofserineorthreoninesometimesarepre
sentinfungalproteins(Nunbergetal.,1984)
Theglycosylationofaproteincanbedifferentdependingonfactorssuchas
themediuminwhichthecellsaregrown.Theglycosylationinfluencesthereac
tionkinetics(iftheproteinisanenzyme),solubility,serumhalf-life,thermalstability,invivoactivity,immunogenicityandreceptorbinding
.Withregardtopeptides,galactosylatedenkephalinsare1000–
10,000timesmoreactivethanthepeptidealone(Warren,
1990).Thatglycosylationincreasesthestabilityofproteins,isshownby
cloninggenesencodingbacterialnon-glycosylatedproteinsinyeast.Theyeastversionswereglycosylatedandmor
estable(Dixon,1991).Glycosylationalsoaffectspharmacokinetics(residen
cetimeinvivo)
(JenkinsandCurling,1994).Examplesofstabilityenhancementaretheprotec
tionagainstproteolyticattackbyterminalsialicacidonerythropoietin(EPO
)(Goldwasseretal.,1974),TissuePlasminogenActivator(TPA)
(WittwerandHoward,1990)andinterferons(Cantelletal.,1992).Withregard
toactivity,humanEPOis1000-foldmoreactiveinvivothanitsdesialylatedformbuttheybothhavesimilarinvi
troactivities(Yamaguchietal.,1991
).Glycosylationoccursthrough(i)anN-
glycosidicbondtotheR-groupofanasparagineresidueinasequenceAsn-X-
Ser/Thr;or(ii)anO-glycosidicbondtotheR-groupofserine,threonine,hydroxprolineorhydroxylysine.However,thes
eaminoacidsmayonlybepartiallyglycosylatedorunglycosylatedleadingto
theproblemofheterogeneity.Inthefuture,clonedglycosyltransferaseswill
beusedtoensurehomogeneity(“glycosylationengineering”)
Methylotrophicyeastshavebecomeveryattractiveashostsforthe
industrialproductionofrecombinantproteinssincethepromoterscontrolli
ngtheexpressionofthesegenesareamongthestrongestandmoststrictlyreg
ulatedyeastpromoters.Thecellsthemselvescanbegrownrapidlytohighden
sities,andthelevelofproductexpressioncanberegulatedbysimplemanipula
tionofthemedium.Methylo-trophicyeastscanbegrowntoadensityashighas130g/
L(Gellisonetal.,1992).Thefourknowngeneraofmethylotrophicyeast(Han
senula,Pichia,Candida,andTorulopsis)shareacommonmetabolicpathwayt
hatenablesthemtousemethanolasasolecarbonsource.Inatranscriptionall
yregulatedresponsetomethanolinduction,severaloftheenzymesarerapid
lysynthesizedathighlevels
ThemajoradvantageofPichiaoverE.coliisthattheformeriscapableofpro
ducingdisulfidebondsandglycosylationofproteins.Thismeansthatincases
wheredisulfidesarenecessary,E.colimightproduceamisfoldedprotein,w
hichisusuallyinactiveorinsoluble.Comparedtootherexpressionsystemss
uchasS2-cellsfromDrosophilamelanogasterorChineseHamsterOvary(CH0)cells,Pichi
ausuallygivesmuchbetter
yields.Celllinesfrommulticellularorganismsusuallyrequirecomplex(rich) media,therebyincreasingthecostofproteinproductionprocess.Additiona lly,sincePichiacangrowinmediacontainingonlyonecarbonsourceandonenitr ogensource,itissuitableforisotopiclabellingapplicationsine.g.proteinNM R.AnadvantageofthemethylotrophP.pastoris,ascomparedtootheryeastsi nmakingrecombinantproteins,isitsgreatabilitytosecreteproteins.Succes shasbeenachievedingeneticallyengineeringtheP.pastorissecretorypathw
aysothathumantypeN-glycosylatedproteinsareproduced(Choietal.,2003).Amongtheadvantageso fmethylotrophicyeastsoverS.cerevisiaeasacloninghostarethefollowing: (i)higherproteinproductivity;(ii)avoidanceofhyperglycosylation; (iii)growthinreasonablystrongmethanolsolutionsthatwouldkillmostothe rmicroorganisms,
(iv)asystemthatischeaptosetupandmaintain,and(v)integrationofmulticop iesofforeignDNAintochromosomalDNAyieldingstabletransformants(G ellisonetal.,
1992)
GlycosylationislessextensiveinP.pastoristhaninS.cerevisiae(Daleetal.,1
999 )duetoshorterchainlengthsofN-linkedhigh-mannoseoligosaccharides,usuallyupto20residuescomparedto50–
150residuesinS.cerevisiae.P.pastorisalsolacksα-1,3- linkedmannosyltransferasewhichproducesα-1,3-linkedmannosylterminallinkagesinS.cerevisiaeandcausesahighlyantigeni cresponseinpatients.Hirudin,athrombininhibitorfromthemedicinalleech ,Hirudomedicinalisisnowmadebyrecombinantyeast(Sohnetal.,2001).Prod uctivitiesofhirudinindifferentsystemsareshowninTable4
P.pastorisproduceshighlevelsofmammalianrecombinantproteinsinth eextracellularmedium.Aninsulinprecursorwasproducedat1.5g/ L(Wangetal.,2001).Otherreportsinclude4g/
Lofintracellularinterleukin2as30%ofprotein,4g/
Lofsecretedhumanserumalbumin(Creggetal.,1993),6g/
Loftumornecrosisfactor(Daleetal.,1999)andotherheterologousproteins(M acauly-Patricketal.,
2005),and10g/
Loftumornecrosisfactor(Sreekrishanaetal.,1989).Productionofserumalb umininS.cerevisiaeamountedto0.15g/LwhereasinP.pastoris,thetiterwas10g/ L(Nevalainenetal.,2005).GelatinhasbeenproducedinP.pastoris,atover14g/ L(Wertenetal.,
1999).P.pastorisyielded300mg/l/
dayofrecombinanthumanchitinase(Goodricketal.,2001).Intracellulartet anustoxinfragmentCwasproducedas27%ofproteinwithatiterof12g/ L(Clareetal.,
1991).ClaimshavebeenmadethatP.pastoriscanmake20–30g/lof recombinantproteins(Morrow,2007)
Therearehowever,somedisadvantagesofusingPichiaasahostforheterolo gousexpression.Anumberofproteinsrequirechaperoninsforproperfoldin g.Pichiaisunabletoproducesuchproteins.AgroupledbyGerngrossmanage dtocreateastrainthatproducesEPOinitsnormalhumanglycosylationform( Gerngross,2004;Hamiltonetal.,
2006).Thiswasachievedbyexchangingtheenzymesresponsiblefortheyeastt ypeofglycosylation,withthemammalianhomologs.Thus,thealteredglycos ylationpatternallowedtheproteintobefullyfunctionalinhumansandsincet hen,thishumanglycosylationofrecombinantproteinsmadeintheengineer edP.pastorishasbeenshownwithotherhumanproteins
HeterologousgeneexpressioninanothermethylotrophHansenulapoly morphayielded1g/LofintracellularhepatitisBS-antigen(50genecopies/ cell),1.4g/Lofsecretedglucoamylase(4copies/cell),and
Table4 Comparisonofproductivitiesofhirudinbyrecombinanthosts Recombinanthostsmg/LBHKcells0.05
Insectcells0.40 Streptomyceslividans0.25–0.5 Escherichiacoli200–300 Saccharomycescerevisiae40–500 Hansenulapolymorpha1500 Pichiapastoris1500
Trang 6byK.lactis
3.3.Filamentousfungi(molds)
FilamentousfungisuchasA.nigerareattractivehostsforrecombinantD
NAtechnologybecauseoftheirabilitytosecretehighlevelsofbioactiveprote
inswithpost-translationalprocessingsuchasglycosylation.A.nigerexcretes25g/
Lofglucoamylase(Wardetal.,
2006
).Foreigngenescanbeincorporatedviaplasmidsintochromo-somesofthefilamentousfungiwheretheyintegratestablyintothechromoso
meastandemrepeatsprovidingsuperiorlong-termgeneticstability.Asmanyas100copiesofagenehavebeenobserved.Tri
chodermareeseihasbeenshowntoglycosylateinamannersimilartothatin
mammaliancells(Salovourietal.,1987)
Thetiterofagenetically-engineeredbovinechymosin-producingstrainofAspergillusawamoriwasimproved500%byconventiona
lmutagenesisandscreening(LamsaandBloebaum,1990).Itwasthenincrease
dfrom250mg/Lto1.1g/Lbynitrosoguanidinemutagenesisandselectionfor2-deoxyglucoseresistance(Dunn-Colemanetal.,
1991,1993).Transformantscontained5–10integratedcopiesofthe
chymosingene.ProductionofhumanlactoferrinbyA.awamoriviarDNAtec
hnologyandclassicalstrainimprovementamountedto2g/
Lofextracellularprotein(Wardetal.,1995).A.nigerglucoamylasewasmadeb
yA.awamoriat4.6g/
L.Humanizedimmunoglobulinfulllengthantibodieswereproducedandse
cretedbyA.niger.Themono-clonalantibodyTrastazumabwassecretedat0.9g/L(Wardetal.,
2004).RecombinantA.oryzaecanproduce2g/Lofhumanlactoferrin
(Wardetal.,1995)and3.3g/LofMucorrennin(Christensenetal.,
1988).FusariumalkalineproteaseisproducedbyAcremoniumchrysogenu
mat4g/L.Recombinantenzymeproductionhasreached
35g/
LinT.reesei(DurandandClanet,1988).ThefungusChrysosporiumlucknow
ensehasbeengeneticallyconvertedintoanon-
filamentous,lessviscous,lowprotease-producingstrainthatiscapableofproducingveryhighyieldsofheterologous
proteins(Verdoesetal.,2007).DyadicInternationalInc.,thecompanyrespo
nsibleforthedevelopmentoftheC.lucknowensesystem,claimsproteinprod
uctionlevelsofupto100g/Lofprotein
Despitetheabovesuccesses,secretedyieldsofsomeheterologousprotei
nshavebeencomparativelylowinsomecases.Thestrategiesforyieldimprov
ementhaveincludeduseofstronghomologouspromoters,increasedgeneco
pynumber,genefusionswithageneencodinganaturallywell-
secretedprotein,protease-deficienthoststrains,andscreeningforhightitersfollowingrandommuta
genesis.Suchapproacheshavebeeneffectivewithsometargetheterologous
proteinsbutnotwithothers.Hence,althoughtherehasbeenanimprovemen
tintheproductionoffungalproteinsbyrecombinantDNAmethods,thereare
usuallytranscriptionlimitations(Verdoesetal.,1995).Althoughanincrease
ingenecopiesuptoaboutfiveusuallyresultsinanequivalentincreaseinprotei
nproduction,highernumbersofgenecopiesdonotgiveequivalentlyhighlevel
sofprotein.SincethelevelofmRNAcorrelateswiththelevelofproteinproduc
ed,transcriptionisthemainproblem.Studiesonoverproductionofglucoa
mylaseinA.nigerindicatetheproblemintranscriptiontobedueto(i)thesiteofi
ntegrationoftheintroducedgenecopiesand(ii)theavailableamountoftrans-acting
Table5
Advantagesofbaculoviralinfectedinsectcellexpressionsystem
Posttranslationalmodifications
ProperproteinfoldingH
ighexpressionlevelsEas
yscaleup
Safety
Flexibilityofproteinsize
Efficientcleavageofsignalpeptides
Multiplegenesexpressedsimultaneously
regulatoryproteins.Also,heterologousproteinproductionbyfilamen-tousfungiissometimesseverelyhamperedbyfungalproteases.Aspergillusni dulanscontainsabout80proteasegenes(Machida,2002)
3.4.Insectcells Insectcells(Table5 )areabletocarryoutmorecomplexpost-translationalmodificationsthancanbeaccomplishedwithfungi.Theyalsoha vethebestmachineryforthefoldingofmammalianproteinsandaretherefore quitesuitableformakingsolubleproteinofmammalianorigin(Agathos,199
1).Themostcommonlyusedvectorsystemforrecombinantproteinexpressi onininsectsisthebaculovirus.Themostwidelyusedbaculovirusisthenuclea rpolyhedrosisvirus(Autographacalifornica)whichcontainscirculardouble
-strandedDNA,isnaturallypathogenicforlepidopterancells,andcanbegrow neasilyinvitro.Theusualhostisthefallarmyworm(Spodopterafrugiperda)ins uspensionculture.Alarvalculturecanbeusedwhichismuchcheaperthanam ammaliancellculture.Recombinantinsectcellcultureshaveyieldedover200 proteinsencodedbygenesfromviruses,bacteria,fungi,plantsandanimals(K night,1991
).Thebaculovirus-assistedinsectcellexpressionoffersmanyadvantages,asfollows
(i)Eukaryoticpost- translationalmodificationswithoutcomplication,includingphosphor-
ylation,N-andO-glycosylation,correctsignalpeptidecleavage,properproteolyticprocessin
g,acylation,palmitylation,myristylation,amida-tion,carboxymethylation,andprenylation(LuckowandSummers,1988;Mil ler,1988).(ii)ProperproteinfoldingandS–
Sbondformation,unlikethereducingenvironmentofE.colicytoplasm (iii)Highexpressionlevels.Theviruscontainsageneencodingtheproteinpoly hedrinwhichismadeatveryhighlevelsnormallyandisnotnecessaryforvirusr eplication.Thegenetobeclonedisplacedunderthestrongcontroloftheviralp olyhedrinpromoter,allowingexpressionofheterologousproteinofupto30
%ofcellprotein.Productionofrecombinantproteinsinthebaculovirusexpr essionvectorsystemininsectcellsreached
600mg/
Lin1988(MaiorellaandHarano,1988).Recentinformationindicatesthattheb aculovirusinsectcellsystemcanproduce11g/
Lofrecombinantprotein(Morrow,2007 ).(iv)Easyscaleupwithhigh-densitysuspensionculture
(v)Safety;expressionvectorsarepreparedfromthebaculoviruswhichcanatta ckinvertebratesbutnotvertebratesorplants,thusinsuringsafety
(vi)Lackoflimitonproteinsize.(vii)Efficientcleavageofsignalpeptides (viii)Simultaneousexpressionofmultiplegenes(WilkinsonandCox,1998) Insectcellsystemshowever,dohavesomeshortcomings,someofwhichc
anbeovercome.(i)Particularpatternsofpost-translationalprocessingandexpressionmustbeempiricallydeterminedfor eachconstruct
(ii)Differencesinproteinsexpressedbymammalianandbaculovirus-infectedinsectcells.Forexample,inefficientsecretionfrominsectcellsmaybe circumventedbytheadditionofinsectsecretionsignals(e.g.,honeybeemelit tinsequence)
(iii)Improperlyfoldedproteinsandproteinsthatoccurasintracellularaggre gatesaresometimesformed,possiblyduetoexpressionlateintheinfectioncy cle.Insuchcases,harvestingcellsatearliertimesafterinfectionmayhelp (iv)Lowlevelsofexpression.Thiscanoftenbeincreasedwithoptimizationoft imeofexpressionandmultiplicityofinfection
(v)Incorrectglycosylationhasbeenaproblemwithinsectcellsashosts(Bisbe e,1993).Thecompleteanalysisofcarbohydratestructureshasbeenreported
foralimitednumberofglycoproteins.PotentialN-linkedglycosylationsitesareofteneitherfullyglycosylatedornotglycosylate datall,asopposedtoexpressionofvariousglycoformsthatmayoccurinmamm
aliancells.Species-specificortissue-specificmodificationsareunlikelytooccur
3.5.Mammaliancells Mammalianexpressionsystemsareoftenusedforproductionofproteinsr
Trang 7equiringmammalianpost-translationalmodifications.Theuseofmammaliancellculture,chieflyimmo rtalizedChinesehamster
Trang 8eplasminogenactivator(tPA)productionintheearlydaysofthebiopharmace
uticaleffort,i.e.,inthe1980s(Swartz,1996).Theseglycosylatedproteinscould
notbeproducedinE.coliatthattime.CHOcellsconstitutethepreferredsystem
forproducingmonoclonalanti-bodiesorrecombinantproteins.Othercelltypesinclude(i)variousmousem
yelomassuchasNS0murinemyelomacells(AndersenandKrummen,2002),
(ii)SF-9,aninsectcellline,
(iii)babyhamsterkidney(BHK)cellsforproductionofcattlefoot-and-mouthdiseasevaccine,
(iv)greenmonkeykidneycellsforpoliovaccine(Wrotnowski,1998)and(v)h
umancelllinessuchashumanembryonickidney(HEK)cells.NSOisanonsecr
etingsubcloneoftheNS-1mousemelanomacellline.In1997,salesofbiotherapeuticsproducedbycell
culturewere
$3.25billionwhereasE.colibasedbiotherapeuticsamountedto
$2.85billion(Langer,1999).By2006,productionoftherapeuticproteinsbyma
mmaliansystemsreached$20billion(Griffinetal.,
2007)
Mammaliancellculturesareparticularlyusefulbecausetheproteinsareo
ftenmadeinaproperlyfoldedandglycosylatedform,thuseliminatingthenee
dtorenaturethem.Eukaryoticcellsarealsousefulforadditionoffattyacidchain
sandforphosphorylatingtyrosine,threonineandserinehydroxylgroups(Qi
u,1998).Mamma-liancellshavehighproductivityof20–60pg/cell/
day.HumantPAwasproducedinCHOcellsat34mg/
Lwithanoverallyieldof47%.AlthoughproductioninE.coliwasatamuchhigh
erlevel(460mg/
L),recoverywasonly2.8%duetoproductionasinclusionbodiesandlowrenat
urationyields(Dartaretal.,1993).Genesfortheglycosylatedfertilityhormone
s,humanchorionicgonadotropin,andhumanluteinizinghormonehavebe
enclonedandexpressedinmammaliancells.Recombinantproteinproducti
oninmammaliancellsrosefrom
50mg/Lin1986to4.7g/
Lin2004mainlyduetomediaimprovementsyieldingincreasedgrowth(Aldr
idge,2006).Atiterof2.5–3g/
Lproteinin14dayCHOfedbatchshakeflaskculturewasachievedusingFe2(Se
O3)3asioncarrier(Zhangetal.,2006).Anumberofmammalianprocessesarepr
oducing3–5g/Land,insomecases,proteintitershave
reached10g/
Linindustry(Ryll,2008).Arathernewsystemisthatofahumancelllineknown
asPER.C6ofCrucellHollandBV,which,incooperationwithDSMBiologics,w
asreportedtoproduce15g/
L(CocoMartinandHarmsen,2008)andthenlater,26g/
Lofamonoclonalantibody(Jarvis,2008)
Manyantibodieswereproducedinmammaliancellcultureatlevelsof0.7
–1.4g/
L.However,highervalueshavebeenreportedrecently.Forexample,monoc
lonalantibodyproductioninNSOanimalcellsreachedover2.5g/linfed-batchprocesses(ZhangandRobinson,
2005
).Animal-free,protein-freeandevenchemically-definedmediawithgoodsupportofproductionhavebeendeveloped.ThePfi
zerorganizationreportedmonoclonalantibodytitersof2.5–3.0g/Linnon-optimizedshakeflaskexperiments(Yu,2006)
Mammaliansystemsdohavesomedrawbacksasfollows
(i)Poorsecretion.Productionofsecretedforeignproteinsbymammaliancel
lsinthe1990samountedto1to10mg/Lwithspecificproductivitiesof
0.1to1pg/cell/
day(WurmandBernard,1999).Theprocessdurationwas5to10days.Althoug
hhighertitershavebeenreached,acceptablelevelswere10–20mg/L
(ii)Mammalianprocessesareexpensive.Thesellingprices(pergram)ofreco
mbinantproteinswere$375forhumaninsulin,$23,000fortPA,
$35,000forhumangrowthhormone,
$384,000forGM-CSF,$450,000forG-CSF,and$840,000forEPO.Allexcepthumaninsulinweremadeinmammali
ancellcultures(Bisbee,
1993).Themanufacturingofmammaliancellbiopharmaceuticalsinafullyva
lidatedplantrequires2to4milliondollarsperyearincostsofmaterialsespecia
llyformedia,15to20milliondollarsperyearinmanufacturingcosts(includin
goverhead,materialandlabor)and40to60milliondollarstoconstructafacility
of25,000ft2and
tovalidateit.AddedontothisisahugecostforgettingFDAapprov al,includingproofofconsistentperformance,productionofabioactiveprod uct,andlackofcontaminationbyvirusesandDNA.Clinicaltrialsand
Trang 9100milliondollars(Bisbee,1993)
(iii)Mammaliancellprocessesalsohaveapotentialforproductcontaminati onbyviruses(Bisbee,1993)
3.6.Transgenicanimals
Transgenicanimalsarebeingusedforproductionofrecombinantprotein sinmilk,eggwhite,blood,urine,seminalplasmaandsilkwormcocoons.Thusf ar,milkandurineseemtobebest.Foreignproteinscanbeproducedinthemamm aryglandsoftransgenicanimals(Bremetal.,1993).Transgenicanimalssucha sgoats,mice,cows,pigs,rabbit,andsheeparebeingdevelopedasproductionsy stems;someaquaticanimalsarealsobeingutilized.Transgenicmiceproduce
tPAandsheepß-lactoglobulinandtransgenicsheepproducehumanFactorIXintheirmilk.Tr ansgenicsheephavebeendevelopedwhichproducemilkcontaining35g/
Lofhumanα-1-antitrypsin,aserumglycoproteinapprovedintheU.S.foremphysema(Wrig htetal.,1991).tPAhasbeenmadeinmilkoftransgenicgoatsatalevelof3g/ L(Glanz,1992).RecombinanthumanproteinC(ananticoagulant)isproduc edinthemilkoftransgenicpigsattherateof
1g/L/
h(Velanderetal.,1992).Cowsproduce30Lofmilkperdaycontainingproteinat 35g/L;thusthetotalproteinproducedperdayis
1kg.Evenifarecombinantproteinwasonlymadeat2g/
L,theannualproductionpercowwouldbe10kg
Theamountsofmilkproducedbyanimals(L/
year)are8000percow,1000pergoat,300persheepand8perrabbit(Rudolph,19
97 ).Productiontiterswere14g/Lofanti-thrombinIIIingoatmilk,35g/Lofα-
1-antitrypsininsheepmilk,and8g/Lofα-glucosidaseinrabbitmilk;allgeneswerefromhumans.Transgenicexpressi onofforeignmilkproteinshasyieldedtitersashighas23g/
Lalthoughtheusualfigureisabout1g/L.Transgenicsheepproduce5g/ Lofrecombinantfibrinogenforuseasatissuesealantand0.4g/
LrecombinantactivatedproteinC,ananticoagulantusedtotreatdeep-veinthrombosis(Dutton,1996).Humanhemoglobinisproducedinpigsat40
g/L.Transgenicexpressionofforeignnon-milkproteinsisusuallymuchlessthanthatofmilkproteins.However,anexc
eptionisthatofhumanα-1-antitrypsininsheepasmentionedabove(Wrightetal.,1991).Inmostcases,the proteinisasactiveasthenativeprotein.Titersofhumangrowthhormoneinmi lkofmiceare4g/Landthatofanti-thrombinIIIis2g/
L.Productioninmilkismorecost-effectivethanthatinmammaliancellculture.Dairyanimalsproduce1to14g /
Lofheterologousproteininmilkeverydayforthe305daylactationcycleeachy ear.TransgenicgoatsproducetPAwithaglycosylationpatterndifferentfrom thatproducedincellcultureandwithalongerhalflifethannativetPA.Transg enicanimalproductshavebeentestedinhumanclinicaltrialsandnoadverser eactionsorsafetyconcernswerereported(McKownandTeutonico,1999) Humangrowthhormonehasbeenproducedintheurineoftransgenicmic e(Kerretal.,1998)butonlyat0.1–0.5mg/
L.Oneadvantageofusingthebladderasabioreactorinsteadofthemammary glandisthatanimalscanurinateearlierthantheycanlactate.Lactationrequir es12monthsforpigs,14monthsforsheepandgoats,and26monthsforcattle,an dlastsfor2monthsforpigs,
6monthsforsheepandgoats,and10monthsforcattle.Theperiodsbetweenlac tationcyclesare2–
6months.Underhormonetreatment,acowproduces10,000Lofmilkperyear comparedto6000Lofurine
Oneofthenegativepointsinproductionofproteinsbytransgenicanimals isthelengthoftimeneededtoassessproductionlevel.Thistakes3.5monthsin mice,15monthsinpigs,28monthsinsheepand
32monthsincows(Chew,1993).Thecostofupkeepofcowsunder
GoodAgriculturalPracticesis$10,000percowperyear
Theproductionofdrugsintransgenicanimalshasbeenstalledbythedemi seofPPLTherapeuticsofScotlandwhich,withtheRoslinInstitute,clonedDo lly,thesheep(Thayer,2003).Theirattempttoproducealungdrugintransgenic sheepforBayerAGwasstoppedandthecompanywasputupforsale
Trang 10ansgenicanimals,toproducerecombinantproteinssuchasvaccines,lymp
hokinesetc.Theproductionoftransgenictrypano-somesexpressingheterologousproteinshasseveraladvantagesovertransg
enicanimals.Theseinclude(i)stableandpreciselytargetedintegrationintot
hegenomebyhomologousrecombination,
(ii)achoiceofintegrationintoseveraldefinedsites,allowingexpressionofmul
ti-subunitcomplexes,and(iii)easymaintenanceofcellsinasemi-definedmediumandgrowthtohighdensities(N2 ×107m
l−1)
3.7.Transgenicplants
Forrecombinantproteinproduction,useofplants,ascomparedtothatofl
iveanimalsandanimalcellcultures,ismuchsaferandlessexpensive,requiresl
esstime,andissuperiorintermsofstorageanddistributionissues.Infact,plant
expressionsystemsarebelievedtobeevenbetterthanmicrobesintermsofco
st,proteincomplexity,storageanddistribution.Theuseofplantsoffersanum
berofadvantagesoverotherexpressionsystems(Table6).Thelowriskofcont
aminationwithanimalpathogensincludesvirusessincenoplantvirusesha
vebeenfoundtobepathogenictohumans.Anotheradvantageisthatgrowth
onanagriculturalscalerequiresonlywater,mineralsandsunlight,unlikema
mmaliancellcultivationwhichisanextremelydelicateprocess,veryexpens
ive,requiringbioreactorsthatcostseveralhundredmilliondollarswhenpr
oductionisscaleduptocommerciallevels
Someaddedadvantagesofplantsystemsareglycosylationandtargeting,
compartmentalizationandnaturalstoragestabilityincertainorgans.Simpl
eproteinslikeinterferons,andserumalbuminweresuccessfullyexpressedi
nplantsbetween1986and1990.However,proteinsareoftencomplexthree-dimensionalstructuresrequiringtheproperassemblyoftwoormoresubun
its.Researchersdemonstratedin
1989and1990thatplantswerecapableofexpressingsuchproteinsandassem
blingthemintheiractiveformwhenfunctionalantibodiesweresuccessfully
expressedintransgenicplants.Bacteriadonothavethiscapacity.Transgenic
plantshavebeenusedtoproducevaluableproductssuchasβ-D
-glucuronidase(GUS),avidin,laccaseandtrypsin(Hood,
2002)
Transgenicplantscanbeproducedintwoways.Onewayistoinsertthedesir
edgeneintoavirusthatisnormallyfoundinplants,suchasthetobaccomosaicvi
rusinthetobaccoplant.Theotherwayistoinsertthedesiredgenedirectlyintoth
eplantDNA.Potentialdisadvantagesoftransgenicplantsincludepossiblec
ontaminationwithpesticides,herbicides,andtoxicplantmetabolites(Fitzg
erald,2003)
Productswithtitersashighas0.02–0.2%ofdrycellweighthave
beenachieved.Recombinantproteinshavebeenproducedintransgenic
plantsatlevelsashighas14%oftotaltobaccosolubleprotein(phytasefromA.ni
ger)and1%ofcanolaseedweight(hirudinfromH.medicinalis)
(Kusnadietal.,1997).Oilseedrapeplantscanproduceenkephalinandaneurop
eptide(Sterling,1989).Thepeptidegenewasinsertedintothegeneencodingth
enativestorageproteinbyscientistsatPlantGeneticSystems(Ghent,Belgiu
m).By1997,twoproducts,avidinandGUSwerereadyforthemarket.GUSfrom
E.coliwasproducedincornat0.7%ofsolubleseedprotein.ActivehepatitisBva
ccine(hepatitisBsurfaceantigen)wasproducedintransgenic
Table6
Advantagesoftransgenicplantsasproteinexpressionsystems
Costeffective
Canproducecomplexproteins
HighlevelofaccumulationofproteinsinplanttissuesLowrisk
ofcontaminationwithanimal;pathogensRelativelysimpl
eandcheapproteinpurification
Easyandcheapscaleup
Properfoldingandassemblyofproteincomplexes
Posttranslationalmodifications
tobaccoplants.Despitethesesuccesses,commercialproductionofdrugsint ransgenicplantswassloweddownbytheclosingdownofthePPLTherapeutic s(Thayer,2003),aswellastheexitofMonsantocorporationfromthiseffort 4.Conclusions
Microbeshavebeenusedtoproduceamyriadofprimaryandsecondarypr oductstobenefitmankindformanydecades.Withtheadventofgeneticengi neering,recombinantproteinsenteredthemarket,whichradicallychange dthescenarioofthepharmaceuticalindustry(Demain,2004).Throughtheu seofrecombinantDNA,importantgenes,especiallymammaliangenes,could beamplifiedandclonedinforeignorganisms.Thisprovidedadifferentappro
achtocomplexbiologicalproblem- solving.Manyoftheresultantbiopharmaceuticalsarepro-ducedusingtechnologicallyadvancedmicrobialandmammaliancellbiosys
tems.Thesecell-based,proteinmanufacturingtechnologiesoffermanyadvantages,produci ngrecombinantpharmaceuticallyimportantproteinswhicharesafeandava ilableinabundantsupply
Generally,proteinsthatarelargerthan100kDareexpressedinaeukaryoti csystemwhilethosesmallerthan30kDareexpressedinaprokaryoticsystem
.Forproteinsthatrequireglycosylation,mamma-liancells,fungiorthebaculovirussystemischosen.Theleastexpensive,easiest andquickestexpressionofproteinscanbecarriedoutinE.coli.However,thisba cteriumcannotexpressverylargeproteins.Also,forS–
Srichproteins,andproteinsthatrequirepost-translationalmodifications,E.coliisnotthesystemofchoice,asitcannotcarry outglycosylationandremovetheS–
Ssequences.Sometimes,eukaryoticproteinscanbetoxictobacteria.Yeastsa reeukaryotes,havetheadvantageofgrowingtohighcelldensitiesandarethus
suitableformakingisotopically-labeledproteinsforNMR.ThetwomostutilizedyeastsareS.cerevisiaeandP.pas toris.Yeastscanproducehighyieldsofproteinsatlowcost,proteinslargerthan5 0kDcanbeproduced,signalsequencescanberemoved,andglycosylationcanb ecarriedout.Yeastsproducechaperoninstoassistfoldingofcertainproteinsa ndcanhandleS–
Srichproteins.Thebaculoviralsystemisahighereukaryoticsystemthanyeas
tandcancarryoutmorecomplexpost-translationalmodificationsofproteins.Itprovidesabetterchancetoobtains olubleproteinwhenitisofmammalianorigin,canexpressproteinslargertha n50kDandS–
Srichproteins,cancarryoutglycosylation,removessignalsequences,haschap
er-oninsforfoldingofproteins,ischeapandcanproducehighyieldsofproteins.Th ebaculoviralsystemishoweverslowandtimeconsumingandnotassimpleas yeasts.Themostpopulartypeofsystemforproducingrecombinantmammal ianglycosylatedproteinsisthatofmammaliancells.Theycangenerateprotei nslargerthan50kD,carryoutauthenticsignalsequenceremoval,glycosylate andalsohavechaperonins.Someoftheproteinsexpressedinmammaliansyst
emsareFactorVII,factorIX,γ-interferon,interleukin2,humangrowthhormone,andtPA.However,select ionofcelllinesusuallytakesweeksandthecellcultureissustainableforonlyali mitedtime.Overall,39%ofrecombinantproteinsaremadebyE.coli,35%byCH Ocells,15%byyeasts,10%byothermammaliansystemsand1%byotherbacter iaandothersystems(Rader,2008)
Geneticallymodifiedanimalssuchasthecow,sheep,goat,andrabbitsecre terecombinantproteinsintheirmilk,bloodorurine.Manyusefulbiopharmac euticalscanbeproducedbytransgenicanimalssuchasvaccines,antibodies,a
ndotherbiotherapeutics.Similarly,trans-genicplantssuchasArabidopsisthalianaandotherscangeneratemanyrecomb inantproteins,e.g.,vaccines,bioplastics,andbiotherapeutics.Commerciald evelopmentoftransgenicanimalsandtransgenicplantshasbeenslowhowe ver,comparedtotheabovesystems
Molecularbiologyhasbeenthemajordrivingforceinbiopharma-ceuticalresearchandtheproductionofhighlevelsofproteins.Thebiopharmac euticalindustryismultifaceted,dealingwithribozymes,antisensemolecu les,monoclonalantibodies,genomics,proteomics,