Production of recombinant human epidermal growth factor in Pichia pastoris

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Biotechnology

and

Industrial

Microbiology

Production

of

recombinant

human

epidermal

growth

factor

in

Pichia

pastoris

Samira

Eissazadeh

a

_,

_Hassan

_Moeini

b,∗

_,

_Maryam

_Ghandizadeh

_Dezfouli

c

_,

Somayyeh

Heidary

a

_,

_Rubina

_Nelofer

d

_,

_Mohd

_Puad

_Abdullah

a

a_{UniversitiPutraMalaysia,FacultyofBiotechnologyandMolecularScience,DepartmentofCellularandMolecularBiology,Malaysia} b_{UniversitiPutraMalaysia,InstituteofBioscience,Malaysia}

c_{ArakUniversity,FacultyofScience,DepartmentofBiology,Iran}

d_{PakistanCouncilofScientificandIndustrialResearch,LaboratoriesComplex,Lahore,Pakistan}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26May2016 Accepted3October2016

AssociateEditor:GiseleMonteirode Souza

Keywords: Pichiapastoris

Humanepidermalgrowthfactor Artificialneuralnetwork

a

b

s

t

r

a

c

t

Thisstudywascarriedouttoexpresshumanepidermalgrowthfactor(hEGF)inPichiapastoris

GS115.Forthisaim,thehEGFgenewasclonedintothepPIC9Kexpressionvector,andthen integratedintoP.pastorisbyelectroporation.ELISA-basedassayshowedthattheamountof hEGFsecretedintothemediumcanbeaffectedbythefermentationconditionsespeciallyby culturemedium,pHandtemperature.ThebestmediumfortheoptimalhEGFproduction wasBMMYbuffered atapHrangeof6.0and7.0.ThehighestamountofhEGFwithan averageyieldof2.27␮g/mLwasobtainedthroughaninductionoftheculturewith0.5%(v/v) methanolfor60h.Theartificialneuralnetwork(ANN)analysisrevealedthatchangesin bothpHandtemperaturesignificantlyaffectedthehEGFproductionwiththepHchange hadslightlyhigherimpactonhEGFproductionthanvariationsinthetemperature.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Humanepidermalgrowthfactor(hEGF)isa6.2kDa polypep-tide composed of 53 amino acid residues with three intramoleculardisulfide bonds.1 _{Oneofitsmajorbiological} functionsistopromotethegenerationofnewepithelialand endothelialcells,andtostimulatetissuerepairs.2 _Assuch, it has a powerful mitogenic activity, which can speed up thehealingprocessofdamagedtissuesdueto,amongother things,ulcersandwounds.hEGFwasalsofoundtobeeffective inthetreatmentsofwrinkles,agespots,frecklesandacnes.3,4 Traditionally,hEGFispurifiedfrom animalurinethrough a

∗ _{Correspondingauthorat:InstituteofBioscience,UniversitiPutraMalaysia,43400Serdang,Malaysia.}

E-mail:[email protected](H.Moeini).

complicatedpurificationstep.5_{However,theyieldandpurity} ofthe hEGFfrom this process arelow andcould notmeet thedemandfromtheindustries.Geneticengineeringisone approach inwhich pure hEGFcan be potentially produced inalargescale.Overthelast twodecades,hEGFhad been producedinvarioushostsystemsincludingEscherichiacoli,6

Bacillus brevis,7 _{Saccahromyces cerevisiae}8 _{andbaculovirus.}9 _In

E. coli, the yield isnot up to the level appropriatefor the

industrialneeds,ascytoplasmichEGFtendstoforminclusion bodies,whichcanberapidlydegradedbyproteases,leading totheformationofmisfoldedhEGFmolecules.Asaresult,the overall productioncostisincreasedduetotheextra down-streamprocessingstepsrequiredtoreleasethehEGFfromthe

http://dx.doi.org/10.1016/j.bjm.2016.10.017

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

inclusionbodies.Inaddition, thehEGFproducedby prokar-yoticsystemexhibitslowerbiologicalactivitiescomparedto thoseproducedbyeukaryoticsystems.Therefore,eukaryotic expressionsystemsmayholdapromisinghostforthe pro-ductionofactivehEGFifpost-translationalprocessingisreally neededespeciallyinalarge-scaleproduction.

Amongtheeukaryoticexpressionsystems,Pichiamayoffer acheaper alternative host forhEGF production. Pichia

pas-torisiswidelyappliedashostsystemsfortheexpressionsof

manyheterologousproteins.Asamethylotrophicyeast,P.

pas-torisisabletogrowinthe presenceofmethanol;therefore,

methanolcanbeusedaninducerwithoutanytoxiceffects. Inthepresentstudy,thesecretaryexpressionvector(pPIC9K) equippedwithastrongmethanolinduciblepromoter(AOX1) was used to express and secrete hEGF extracellularly.10,11 PichiahasboththeadvantagesofE.coliandeukaryotic expres-sionsystemsforbeinganinexpensive hostsystem, ableto highlyexpressheterologousproteins,andabletomodify pro-teinspost-transnationally.

Materials

and

methods

Microorganismsandplasmids

E.coliDH5␣,asahostforconstructionofDNAplasmids,was cultured in Luria Bertani (LB)medium supplemented with 50␮g/mLampicillin.P.pastorisstrainGS115,asahostforthe productionofhEGF,culturedinYPDmedium[1%(w/v)yeast extract,2%(w/v)peptone,2%(w/v)dextrose(glucose)]at30◦C. DNA plasmid pFLAG-ATS-hEGF, obtained from the Faculty ofBiotechnologyandBiomolecularSciences,UPM,Malaysia, wasusedatemplatefortheamplificationofhEGFgene.The pPIC9Kexpressionplasmid(Invirtrogen,USA)wasusedforthe expressionofhEGFinP.pastoris.

ConstructionofpPIC9K-hEGFDNAplasmid

ThehEGFgene(GeneBankAccessionNo.M15672)was ampli-fied from the previously constructed plasmid pFLAG-ATS-hEGF12 _{using the specific primers 5} -GAATTCATGAACTCA-GATAG-3and5-CCTAGGTCAACGTAATTCC-3.Amplification wascarried out inathermocycler(GeneAmp PCRsystem, BioRad,USA);afterdenaturationat95◦Cfor2min,the sam-plewassubjectedto35cycleof95◦Cfor30s,57◦Cfor30s, and72◦Cfor20sfollowedbyfinalextensionat72◦Cfor5min. TheamplifiedfragmentwastheninsertedintotheEcoRIand

AvrIIcloning sitesofthe yeastexpressing plasmidpPIC9K.

AftertransformationoftherecombinantplasmidsintoE.coli

DH5␣cells,geneinsertionwascheckedbyrestrictionenzyme digestionandPCR.Theintegrityoftheinsertwascheckedby double-strandedsequencing(1stBASEDNASequencing Ser-vices,Malaysia).

ExpressionofhEGFinP.pastoris

Electro-transformationofpPIC9K-hEGFintoP.pastoris

AsinglecolonyofP.pastorisGS115wasculturedin5mLYPD mediumina50mLconicalflaskovernightinashaker incu-batorat30◦C with250rpmagitation rate.Theculturewas

transferredinto freshYPDmedium(250mL) andincubated underthesameconditionuntiltheOD600nmreached1.3–1.5. Thecells werethen harvestedat1500×gfor5minat4◦C; following2timeswashingwith500and250mLice-cold ster-ile dH2O, the cellswere resuspended in20mLice-cold1M sorbitol.Finally,thecellswerecentrifugedandresuspended in1mLice-cold1Msorbitol.Forelectro-transformation,ina 0.2cmcuvette(BioRad,USA),the P.pastoriscompetent cells weremixedwith0.5␮goftherecombinantpPIC9K-hEGF plas-mid;after5minincubationonice,electroporationwascarried outat1.5kV.Immediatelyafterelectroporation,1mLof ice-cold1Msorbitolwasaddedtothecells;inasterilemicrotube, thecellsweremixedwith1mLYPDmediumandincubatedas at30◦Cfor1h.Around200␮LofthemixturewasspreadonMD agarplates[1.34%yeastnitrogenbasewithammoniumsulfate withoutaminoacids(YNB),4×10−5%biotinand2%dextrose]. Theplateswereincubatedat30◦Cuntilrecombinantcolonies wereappeared(2–5days).

Directscreeningofmultipleinserts

PositivetransformantswerescreenedontheYPDagar sup-plementedwithgeneticinatthefinalconcentrationsof0.25, 0.5,0.75,1.0,1.5,1.75,2.0,3.0and 4mg/mL.Positiveclones were screenedbydirectcolonyPCRaspreviouslydescribed byLinderet al.13 _{withsomemodifications.Briefly,asingle} colonyofthetransformantswaspickedandresuspendedin 10␮LofsteriledH2O. Then,5␮Lof 5U/␮Lsolution of lyti-casewasaddedtothecells;after10minincubationat−80◦_C, the extractwas subjectedtoPCRusing the 5 and3 AOX1 primersaccordingtothefollowingcycles:95◦Cfor5minfor pre-denaturation,30cyclesofamplificationat95◦Cfor1min, 54◦Cfor1min(annealing)and72◦Cfor1minfollowedbya finalextensionstepat72◦Cfor5min.

Invivoscreeningofmultipleinsertions

His+_{cloneswereresuspendedindH}

2O.Then,thesuspensions weretransferredto50mLFalcontube,vortexedfor8sandcell densitywasdeterminedat600nmusingaspectrophotometer. ThecellswereplacedonYPDcontaining0.25,0.5,0.75,1.0, 1.5,1.75,2.0,3.0and4mg/mLgeneticin,separately.Theplates wereincubatedat30◦Candmonitoredforsurvivalseveryday.

ProductionofrecombinanthEGF

Ina250mLbaffledflask,asinglecolonyoftherecombinant

P.pastoriswasinoculatedin25mLBMGY[buffered

glycerol-complexmedium:1%(w/v)yeastextract,2%(w/v)peptone, 100mMpotassiumphosphate,pH6.0,1.34%(w/v)yeast nitro-genbase(YNB),4×10−5%(w/v)biotin]containing1%glycerol (v/v)followedbyovernightincubationat30◦Cinashaking incubator(250rpm)untiltheopticaldensityat600nmreached 2(log-phasegrowth).Thecellswerethenharvestedat2500×g

for5minatRT;thepelletwasresuspendedin150mLofBMMY mediumsupplemented with0.5%methanol andincubated untiltheopticaldensityat600nmreached1.0.Ina1Lbaffled flask,theculturewasfurtherincubatedat29◦Cwith agita-tionat270rpmfor96h.Inordertomaintaintheinduction, methanolwas addedtoafinalconcentrationof0.5%every 24h.TheculturewascheckedforhEGFproductionat0,6,12, 24,36,48,60,72,84and96hofincubation.

SDS-PAGEanalysis

Secreted proteins were separated on a 12% SDS-polyacrylamide gel. Eighty micro-liter of each sample was boiled in a 20␮L sample buffer (312mM Tris–HCL pH 6.8, glycerol50%(v/v),bromophenolblue0.05%(w/v)anddH2)for 10minbeforethesamplemixturewerebeingloadedontothe gel.StandardhEGFprotein(65.0kDa)(R&DSYSTEM)wasused asapositivecontrol.hEGFproteinwaspurifieddirectlyfrom the SDSgel usinga commerciallyavailablepolyacrylamide gelproteinpurificationkit(ThermoScientific).

QuantikinehEGFimmunoassay

For hEGF expression assay, the Quantikine Human EGF immunoassaykit(R&Dsystem,USA)wasusedaccordingto themanufacture’sinstruction.Astandardcurvewascreated byplottingthemeanabsorbanceat570nmforeachstandard

onthe y-axis againstthe concentrationon the x-axis. The

amountofhEGFwasdeterminedbyreferringtheabsorbance valueofthesamplesat570nmtothehEGFstandardcurve.The datawerelinearizedbyplottingtheEGFconcentrationsversus thelogoftheabsorbanceandthebest-fitlinewasdetermined byregressionanalysis.

EffectofcultureconditionsongrowthandhEGF production

Culturemedium

In1-Lbaffledflask,thecellsweregrownon150mLofYPD, BMMY,MMorBMGY.CellbiomassandhEGFproductionwere determinedat0,12,24,36,48,60,72and84hafter cultur-ing.Thegrowthconditionusedinthisexperimentwassimilar withtheonedescribedearlier.Methanolwasusedatafinal concentrationof0.5%(MMandBMMY)every24hinorderto maintainacontinualinductionand its finalconcentration. Thebest mediumwas selectedforfurther optimizationas below.

Methanolconcentration

Therecombinantsweregrowninthesameconditionas pre-viouslydescribed. Differentconcentrations ofmethanol [0, 0.25, 0.5, 1, 1.5 and 2% (v/v)] were evaluated. The respec-tive final concentrations ofmethanol were maintained by addingtheappropriateamountsofmethanoltothecultures forevery24h.Samplingsoftheculturesweredoneevery12h asdescribedabove.

pH

Optimizationwasdoneintwosteps;inthefirststep,alarge rangeofpHfrom 4to7was used; inthesecond step,the pHrangewasfurtherfine-tunedbased onthetwobest pH obtainedfromthefirststep.Thecultureswereincubatedusing the same cultureconditionthat wasdescribed before. The samplesweretakenevery12hforanalysis.

CombinationofpHandtemperature

TherangeofpHusedinthisstudywasbetween4and7 com-prisingfourdifferentlevelsofpHsi.e.4.0,5.0,6and7;whereas theeffectoftemperaturewasevaluatedat25◦C,30◦C,32◦C and35◦C.Atotalofsixcombinationswereperformedandthe growthconditionusedwasthesamewiththeonementioned

before.Theartificialneuralnetwork(ANN)wasusedto opti-mizepHandtemperatureforhEFGproduction.WeusedANN withthreelayers:(1)aninputlayerwith2neurons,(2)ahidden layerwith7neurons,and(3)anoutputlayerwith1neuron. Thetopologyofthenetworkcontainedthreelayers(2:7:1):one inputlayerfortwofermentationvariables,pHand tempera-ture;middlehiddenlayerof7neurons,andoneoutputlayer forhEGFproduction.

Statisticalanalysis

Thedatawereanalyzedbyusingonewayanalysisofvariance followedbylinercorrelationtestswhereapvalueoflessthan 0.05wasconsideredassignificant.Thestatisticalanalysisof theANNdataandplotswereperformedusingtheStatistical softwareversion7.

Results

ConstructionofpPIC9K-hEGFexpressionplasmid

ThehEGFgenewasamplifiedfromthepFLAG-ATS-hEGF plas-mid and inserted into the expressionvector pPIC9K. After transformationintoE.coliDH5␣,positivecloneswereselected withdirectPCRscreeningandrestrictionenzymedigestion. Finally, theidentity ofthe insertswere checkedby double-stranded sequencing pPIC9K-hEGF plasmid was sequenced andcomparedtotheoriginalhEGFsequence.The compari-sonanalysisshowedtheclonedhEGFwas100%similartothe originalhEGF(GeneBankAccessionNo.M15672).

GenerationofrecombinantP.pastoris

TheDNAplasmidconstructpPIC9K-hEGFwaslinearizedby

SacIandelectro-transformedintoP.pastoris.Therecombinant cloneswere screenedon YPDsupplementedwithdifferent concentrationsofgeneticinfrom0.25to4mg/mL.Accordingto theresults(datanotshown),mostofthetransformantswere foundtoberesistanttothelowconcentrationofGeneticin (0.25,0.5and0.75mg/mL)andjustafewwereresistanttothe highconcentrations.DirectPCRprovedsuccessfulinsertionof

thehEGFgeneintoP.pastorisgenome(Datanotshown).

ProductionofrecombinanthEGF

Five recombinant clones selected from the hyper-resistant clones were tested for the production of hEGF by ELISA at ten different time points after induction. As shown in Table1,thehighestlevelofhEGFproductionwasobtained60h post-induction.Veryweakabsorbancereadingsconsideredas backgroundreadingoftheELISAassaywereobservedinthe controlsample(GS115). BothB1and D7clonesshowed the highesthEGFproduction,60hpost-induction,however,inD7, thelevelofhEGFwasfoundtobedecreasedrapidly.Therefore, theB1clonewasselectedfortheoptimizationoffermentation conditionsandmediumcompositions.

Based on the highestlevel ofhEGF protein production, thepresenceofhEGFintheculturefiltratewasfurther ana-lyzedbySDS-PAGE,asshowninFig.1.ThehEGFproteinband

Table1–hEGFproductionindifferentclonesofP.pastoris.

Time(h) hEGF(␮g/mL)

GS115(control) RecombinantP.pastorisstrains

D7 A5 A8 B1 C2 0 0 0 0 0 0 0 6 0 0 0 0 0 0 12 0 0 0 0 0 0 24 0 0.44±0.26 0.20±0.1 0.27±0.40 0.63±0.60 0.05±0.30 36 0.01±0.25 0.76±0.15 0.42±0.21 0.44±0.25 0.89±0.15 0.08±0.11 48 0.03±0.31 1.31±0.21 0.62±0.17 0.64±0.21 1.31±0.15 0.18±0.21 60 0.06±0.28 2.25±0.15 0.89±0.21 0.89±0.40 2.27±0.21 0.4±0.25 72 0.08±0.41 2.1±0.10 0.86±0.23 0.83±0.36 2.20±0.10 0.36±0.2 84 0.05±0.28 1.85±0.05 0.84±0.21 0.77±0.30 2.11±0.17 0.31±0.17 96 0.04±0.45 1.61±0.25 0.79±0.25 0.67±0.25 2.00±0.25 0.26±0.25

representedtheexpectedsizeoftheprotein(6.2kDa)as

com-paredtothestandardhEGFandthemarker.Thetotalamount

ofsecretedproteinsinthemediumwasalsodeterminedby

theBradfordassay,where10.4mg/mLproteinwasdetected

fortheB1strain.ThehEGFproteinwasthenpurified.

GrowthcurveandhEGFproduction

ThegrowthcurveoftherecombinantP.pastorisshowedashort

lagphase,lastingforafewhours,followedbythe

exponen-tialgrowthfor50h,beforereachingthestationaryphase10h

later(Fig.2).ThefirstappearanceofhEGFwasobservedafter

24hofculturei.e.shortlyafterbeinginducedat20h.Arapid hEGF production occurred concomitantly with an increase inthe growth.Under this condition, the hEGFwas contin-uallyproducedevenaftertheculturereacheditsstationary phase.

OptimizationofcultureconditionforhEGFproduction

Baffledflaskwasusedforoptimizationoftheculturecondition forgrowthandhEFGproductioninP.pastoris.

M 200 KDa 55 KDa 14 KDa 6 KDa 6.2 KDa 1 2 3 4

Fig.1–SDS-PAGEoftotalproteinsecretedfromthewild andrecombinantstrainofP.pastoris.TheSDSgelwas stainedbyCoomassineblue.M:stainedproteinladder; lane1:standardhEGFprotein;lanes2and3:samplesof recombinantP.pastorisB1clone;lane4:sampleofwild

typeP.pastorisGS115.Eachwellwasloadedwith100␮Lof

culturefiltratefromrecombinantP.pastorisB1andwild

typeP.pastoris.

Culturemedium

AtimecourseinvestigationofhEGFproductionandbiomass accumulationindifferentmediainshakeflaskswascarried outwherethehighesthEGFproductionwasobtainedinBMMY andMMmedium,asshowninFig.3.Intermofbiomass pro-duction,allofthemediawerefoundtosupportgoodgrowth oftheculture.Although,thebestcellbiomassproductionwas observedintheYPDmedium,thismediumfailedtopromote goodproductionofhEGF.

Methanolconcentration

The effect of different concentrations of methanol (0.0%, 0.25%,0.5%,1.0%, 1.5%and 2%) ongrowth ofthe

recombi-nantP.pastorisandhEGFproductionarepresentedinFig.4A

andB.AdecreaseintheproductionofhEGFandcellbiomass wasdetected,whentheconcentrationofmethanolwaseither toolowortoohigh,althoughthegrowthratewasfoundto behigheronlyatrelativelylowconcentration(0.25%ofthe finalconcentration).ThehighestlevelofhEGFproductionwas observedwhentheconcentrationofmethanolwas0.5%(v/v).

pHandtemperature

AsshowninFig.5,higheryieldofhEGFwasdetectedatapH rangeof6–7.EventhoughPichiacangrowonapHrangeof4 and5,whichisslightlyacidic,theamountofhEFGwaslow. Fig.6showstheeffectofanarrowpHrangeonhEGF produc-tion.Theresultalsoshowedthattherecombinantscouldgrow

3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 20 40 60 80 100 Time (h)

hEGF protein (ug/ml)

Dr y cell (mg/ml) 0 10 20 30 40 50 60 70 80 90 100

Fig.2–AtimecourseofgrowthandhEGFproductionby therecombinantP.pastoris.Dataaremeansofbiological triplicatereadings.Drycell( ),hEGFprotein( ).

100

A

B

90 80 70 60 50 40 30 20 10 0 0 20 40 60 Time (h) Time (h) BMGY BMGY BMMY BMMY MM _MM YPD YPD Dr y w e ight of cell biomass (mg/ml)

hEGF protein (ug/ml)

80 100 0.0 24 36 48 60 72 84 0.5 1.0 1.5 2.0 2.5

Fig.3–Atime-coursegrowthandhEGFproductionbytherecombinantP.pastorisondifferentmedia.(A)Growthcurvesof therecombinantP.pastorisondifferentmedia.(B)hEFGproductionondifferentmedia.Dataaremeansofbiological triplicatereadings. 3.0

A

B

100 90 80 70 60 50 40 30 20 10 0 0 20 40 60 80 2.5 2.0 1.5 1.0 0.5 0.0 24 36 48 60 Time (h) Time (h) 0% M 0.25% M 0.50% M 1% M 1.5% M 2% M

hEGF protein (ug/ml)

Dr

y w

eight of cell biomass (ug/ml)

72 84

Fig.4–Atime-coursehEGFproductionandcellbiomassofrecombinantP.pastorisafterbeinginducedbydifferent concentrationsofmethanol.(A)EffectsofdifferentmethanolconcentrationsonhEGFproduction.(B)Effectsofmethanol concentrationsongrowthoftherecombinantyeast.Dataaremeansofbiologicaltriplicates.0%methanol( ),0.25% methanol( ),0.5%methanol( ),1%methanol( ),1.5%methanol( )and2%methanol( ).

3.0

A

B

100 90 80 70 60 50 40 30 20 10 0 2.5 2.0 1.5 1.0 0.5 0.0 24 36 48 Time (h) Time (h) pH4 pH5 pH6 pH7 Dr y w

eight of cell biomass (mg/ml)

pH4 pH5 pH6 pH7

hEGF protein (ug/ml)

60 72 84 0 20 40 60 80 100

Fig.5–EffectsofpHoncellbiomassandhEGFproductionbytherecombinantP.pastoris.(A)EffectofpHonhEGF production.(B)EffectofpHongrowthoftherocombinantculture.Dataaremeansofbiologicaltriplicates.

inawidepHrangefrom3.0to7.0,howeverthegrowthratewas foundtobeloweratpHbelow6.0.

Data ofthe combination effects ofpHand temperature analyzedbyANN,asshown inTable 2, revealedsignificant effects of both pH and temperature on hEGF production. Theoptimum pH and temperature calculated bythe ANN were found to be 7 and 29◦C, respectively. The predicted amountofhEGFproductionattheselevelswas2.371␮g/mL. The combinatorial effects of pH and temperature were

representedbythesurfaceplotwherechangesintheculture mediapHshowedmoreimpactonthehEGFproductionthan temperature (Fig. 7A). Theanalysis ofANN sensitivitywas carriedouttoanalyzetheratioandrankingofthevariables wherevariableswitharatiohigherthanonewereconsidered assignificant.14_{Basedonthis,bothparameterssignificantly} influenced the production of hEGF (Table 3). Theeffect of individual factor on hEGF production is shown in Fig. 7B and 7C. The productionof hEGF was increased as the pH

Table2–EffectofdifferentpHandtemperatureonhEGFproduction. Time(h) hEGF(␮g/mL) 25◦_{C 30}◦_C pH 4 5 6.5 4 5 6.5 0 0 0 0 0 0 0 12 0 0 0 0 0 0 24 0 0 0.45 0.09 0.25 0.27 36 0 0.21 0.78 0.11 0.45 1.05 48 0.07 0.25 1.35 0.22 0.49 1.62 60 0.11 0.40 2.21 0.26 0.65 2.48 72 0 0.19 2.17 0.18 0.44 2.44 84 0 0.05 2.10 0.12 0.30 2.37 3.0 pH5.5 pH5.75 pH6 pH6.25 pH6.5 pH6.75 pH7 2.5 2.0 1.5 1.0 0.5 0.0 24 36 48 60 Time (h)

hEGF protein (ug/ml)

72 84

Fig.6–Atime-courseofhEGFproductionbythe

recombinantP.pastorisinanarrowrangeofculturemedia pH.Dataaremeansofbiologicaltriplicates.

Table3–SensitivityanalysisbyANN.

pH Temperature

Ration 1.994860 1.696055

Rank 1.000000 2.000000

increasedhigherthan4.0.TheoptimaltemperatureforhEGF

productionwas29◦CandthelevelsofhEGFwasexpectedto

bedecreasedwhenthetemperaturewashigherthan31◦C.

Discussion

ThisstudywascarriedouttoproducerecombinanthEGFin

P. pastorisGS115usinga commerciallyavailableexpression

vectorequippedwithaspecificsignalpeptidethatdirectsthe

transportationofthehEGFproteinintotheculturemedium.

TheabilityofP.pastoristousemethanolasasolecarbonand

energysourceallowstheuseofmethanolalcoholoxidasegene

(AOX1) promoterforthe expressionofthe desiregene.15,16

Glycerolwasusedasasolecarbonsourceuntiltheyreached thelog-phasegrowthwheremethanolwassupplementedin themediumforhEGFinductionassuggested.17

ThehEGF genewasinsertedinto thepPIC9Kexpression vector,andthenintegratedintotheyeastbyelectroporation.

ThecloneswerescreenedonYPDsupplementedwith differ-ent concentrations ofgeneticin from 0.25 to4mg/mL.The level of geneticin resistance depends on the copy number of the kanamycinegene integrated into the Pichia genome and itseemsthat multipleintegrationsofthe recombinant

gene in the Pichia genome increase the expression of the

desired protein.18–21 _{Therefore, clonesresistant tothe high} concentrationofgeneticin wereselectedfortheproduction ofrecombinanthEGF.

ELISA-basedassayconfirmedsuccessfulsecretionofhEGF intheBMMYmedium.HighestlevelofhEGFwasdetectedin thehyper-resistantcloneB1,60hpost-induction.

TheamountofhEGFsecretedintothemediumwasshown tobeaffectedbythefermentationcondition.Optimizationof cultureconditions isoneofthe determiningfactorsto the successofrecombinantproteinproductioninyeaststrains. Shakenflask,small-scaleexpressionmethodsareoftenthe first step in order to optimize the protein expression lev-elsandselectingthebestcultureconditions,22_althoughthe levelofproteinproductionfromshakeflaskculturesis10-fold lowerthanthelevelthatwouldbeachievedusingafermenter becauseofitslowercelldensity23_{andlimitedaeration.}24,25

Highest level of hEGF was detected in BMMY and MM medium.Itcanbeduetothepresenceofmethanolinthese mediawhichactsasolesourceofcarbonandenergyaswell asaninducerforthegeneexpressionsystem.Itisacommon practiceforhighproductionofrecombinantprotein,high den-sitiescultureisusedtopromotehighyieldproteinproduction basedonthefactthatthelevelofrecombinantprotein pro-duction isproportionalto celldensity.11 _{However,this was} nothappenedinthisstudy,asYPDmediumwhichpromoted goodgrowthoftheyeastculturefailedtoinducehEGF pro-duction.BMMYwhichsupportgoodgrowthandhighestlevel ofhEGFproductionwasselectedasthecultivatingmedium fortherecombinantB1strain.Thismediumhasbeenshown tobealsothebestmediumfortheexpressionofSAG2and bikunin.26,27

OptimizationofmethanolconcentrationinPichia fermen-tationsystemisextremelyimportantbecauseahighlevelof methanolcanbetoxictotheyeast.28_{Insomecases,increasing} methanolconcentrationcausesincreasingintheexpression levelsuchasexpressionofhuman␤-2-glycoproteinI.29 How-evertheaccumulationofmethanolhasanegativeresultofcell growthandcanleadtoadecreaseintheexpression.25,30_On

–0.5 36 34 32 30 28 pH

B

A

C

2.5 2 1.5 1 0.5 0 –0.5 Te_mper ature Production 26 24 3.5 4.0 4.5 5.0 5.56.0 6.57.0 7.5 0.0 0.5 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 –0.2 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 23 24 25 26 27 28 29 30 Temperature 31 32 33 34 35 36 5.4 5.6 pH Production 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Production 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 1.0 1.5 2.0 2.5 3.0

Fig.7–(A)Three-dimensional(3D)surfaceplotshowingtheeffectofpHandtemperatureonhEGFproduction.(B)One-factor contoureplotshowingtheeffectofpHonhEGFproduction.(C)One-factorcontoureplotshowingtheeffectoftemperature onhEGFproduction.

theotherhand,alowlevelofmethanolmaynotbeenoughto initiatethetranscriptionoftherecombinantgene.10_Methanol concentration of0.5% (v/v)was considered as the optimal concentrationfortheproductionofhEGFwhichissimilarto whathasbeenreportedforlaccaseproductioninP.pastoris

hostsystem.31_{Itwasshownthatmethanolwith} concentra-tionhigherthan0.5%(v/v)mightinhibitgrowthwhereasvery lowconcentrationofmethanolmightnotbeenoughto sup-portenergymetabolismleadingtopoorgrowthofcultures. Infact,methanolwasnotonlyusedtoprovideasolecarbon sourceforPichiabutalsotoinducehEGFproduction.However, thelevelsofmethanolshouldbekeptlowtoavoidthetoxicity effectofmethanol,32_{whichwouldinhibitcellgrowthand} pro-teinproduction.Thetoxicityeffectofmethanolwasevident at1%(v/v)withthepoorgrowthrateoftheculture,whichin turnreducedthehEGFproduction.

Liketemperature,pHofculturemediaplaysanimportant roleforoptimizedheterologousproteinproductionasitisnot onlyaffecttheyieldofheterologousproteinproductionbut alsoaffecttheintegrationoftheproteinstructure.33_In addi-tion,thepHofculturemediawouldalsoaffecttheactivities

ofvariousproteasesthatmaydegradeheterologousproteins. Ourresultsshowedthat therecombinantPichiacouldgrow inawidepHrangefrom3.0to7.0,althoughhighestyieldof hEGFwasobtainedatthepHrangeof6–7.Thisfeatureisan extraadvantageoftheyeastforrecombinantprotein produc-tionwhereawiderangeofpHcanbeusedfortheoptimization oftherecombinantproteinproductionasdemonstratedinthis study.Data analysisbyANNshowed thesignificant effects ofbothpHand temperatureon hEGFproduction,although changesinpHwerefoundtobemoreeffective.ANN analy-sisrecommended29◦CastheoptimaltemperatureforhEGF productioninP.pastorisandtheyieldofhEGFwasexpectedto bedecreaseattemperatureshigherthan31◦C,possiblydueto poorstabilityoftheproteinathighertemperatures,releasing ofproteasesfromdeadcells,andmisfoldingoftheprotein.34,35 In conclusion, this study supportsthe use ofP. pastoris

asahostforhEGFproduction,specificallywhentheoverall hEGFproductioniscomparedwiththeproductioninE.coli.

TheBMMYmediumwithpH7supplementedwith0.5%(v/v) methanolisrecommendedasthebestinductionmediumfor hEGFproductioninP.pastoris.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbythefacultyofBiotechnologyand BiomolecularSciences,UniversitiPutraMalaysia,Malaysia.

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