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A Thesi
s
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heDegr
eeofMast
erofSci
ence
Pr
ocessopt
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orpr
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ecombi
nantPED ant
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수용성 재조합 PED 항원 단백질 생산을 위한
공정기술 최적화
August
,2015
By
DoWoon Shi
n
Depar
t
mentofAgr
i
cul
t
ur
alBi
ot
echnol
ogy
Gr
aduat
eSchool
농 학 석 사 학 위 논 문
Pr
ocessopt
i
mi
zat
i
on f
orpr
oduct
i
on ofsol
ubl
e
r
ecombi
nantPED ant
i
gen
수용성 재조합 PED 항원 단백질 생산을 위한
공정기술 최적화
지도교수 최 윤 재
이 논문을 농학 석사학위논문으로 제출함
2015년 7월
서울대학교 대학원 농생명공학부
신 도 운
신도운의 농학 석사학위논문을 인준함
2015년 7월
위
원
장
(
인)
부 위 원 장
(
인)
위
원
(
인)
Summar
y
Porcine epidemic diarrhea (PED) is a contagious disease and
causessignificanteconomiclossesinmostoftheswinefarmsin
korea.TopreventPED,arecombinantE.coliexpressing aPED
subunitvaccinewaspreviously developedby ourresearch group.
E.coli is the most widely used host for the production of
recombinantproteins because itis well-characterized system in many aspects. In order to develop efficient productivity of vaccines,anincreaseofE.colicelldensity wasfocusedforhigh yield production. The influence of expression temperature, induction time, harvest time conditions and concentration of inducersincluding IPTG (isopropyl-1-thio-â-D-galactopyranoside)
and lactose on soluble expression ofPED subunitvaccine was
investigated. In the shake flask culture condition, pTf16
chaperone co-expression,expression temperature 15℃,0.1 mM
IPTG and1mM lactoseasinducer,inductionOD6000.6and
harvesttime 12 h (rGST-COE),24 h (rGST-S1D)were most
favorable conditions for the highest protein expression. The concentrationofpurifiedproteinwas5mg/linflaskculturelevel.
The result of western blot confirmed the expression of
rGST-COE andrGST-S1D.
Batch fermentation was performed with synthetic media using
the screened pTf16 chaperone co-expression, expression
temperature 15℃ and 0.1 mM IPTG.In the fermenter culture
condition,induction OD600 3∼4, growth temperature 30℃, 0.5
mg/ml L-arabinose were most favorable conditions for the
enhanced by around 10 times from flask level (5 mg/l) to fermentation level(53.8 mg/l).The amino acid sequence ofthe
purifiedproteinwasidenticaltothatofrGST-COE,showingthat
optimized fermentation and purification system enable highly efficientexpressionofPED subunitvaccineinaccuracy.
Theseresults showed thattheoptimized fermentation condition forsoluble expression ofPED vaccine can enhance the efficacy
ofmass-productionofPED vaccine.
Key words: Chaperone, Condition-optimization, E. coli,
Fermentation, IPTG, L-arabinose, Lactose, PED, Purification, Solubleproteinexpression
Cont
ent
s
Summary I
Contents III
ListofTablesandFigures VI
ListofAbbreviations Ⅸ
I.Introduction 1
II.Review ofLiterature 3
1.Porcineepidemicdiarrheavirus 3
1)CharacteristicsofthePEDV 3
2)PEDV spikeprotein 5
2.Strategies to optimize mass-production soluble protein
expression 6
1)Chaperoneco-expression 6
2)Modificationofthecultureconditions 8
(1)Theeffectoftemperature 8
(2)Theeffectofinducerconcentration 9
(3)Theeffectofinductiontime 10
III.MaterialsandMethods 11
1.Optimization offlask cultureconditions 11
1)Strainsandmedia 11
2)Cultureconditions 12
3)Chaperoneco-expressionscreening 13
4)Sonication 13
5)SDS-PAGE 13
6)Westernblot 14
8)OptimizationofIPTG concentration 14
9)Optimizationofinductiontime 15
10)Optimizationofharvesttime 15
11)ComparisonofIPTG withlactoseasinducer 15
12)PurificationofrGST-COE from solublefraction 15
2. Development of fermentation production process and
antigen purification 17
1)Strainandmedia 17
2)Cultureconditions 17
3)Optimizationofgrowthtemperature 18
4)Optimizationofinductiontime 18
5)OptimizationofL-arabinoseconcentration 19
6)PurificationofrGST-COE from solublefraction 19
IV.ResultsandDiscussion 21
Chapter1.Optimization offlaskcultureconditions 21
1.Chaperoneco-expression 21
1)Chaperoneco-expressionscreening 21
2)Confirmationofchaperoneeffect 24
2.Confirmation ofprotein expression by western blot 26
3.Modification ofthecultureconditions 28
1)Optimizationofexpressiontemperature 28
2)OptimizationofIPTG concentration 30
3)Optimizationofinductiontime 32
4)Optimizationofharvesttime 34
5)ComparisonofIPTG withlactoseasinducer 36
4.Purification ofrGST-COE from solublefraction 38
Chapter 2.Developmentoffermentation production process
andantigen purification 39
1.Monitoring growth curve 39
1)Comparisonofculturemedium forfermentation 39
(1)BetweenLB brothandsyntheticmedium 39
2)Comparisonofgrowthtemperatureforfermentation 41
(1)Betweengrowthtemperature30℃ and37℃ 41
2.Optimization ofbatch typeexpression 43
1) Optimization of growth temperature 30℃ and 37℃ at
inductionOD6007∼8 43
2) Optimization of growth temperature 30℃ and 37℃ at
inductionOD6003∼4 45
3)Optimization ofinduction OD6003∼4 and 7∼8 atgrowth
temperature30℃ 47
4)OptimizationofL-arabinoseconcentration 49
3.Purification ofrGST-COE from solublefraction 52
1)GSTrapcolumnmethod 52
4. Electrospray ionization assay of purified rGST-COE
protein from solublefraction 54
V.Conclusion 56
VI.LiteratureCited 59
Li
stofTabl
esandFi
gur
es
Tabl
es
Table1.Plasmidsusedinthisstudy. 11
Table2.Chaperoneteam codedoneachplasmidandinducer. 12
Table3.Buffersforcolumnscreening. 20
Table4.ProcedureofGSTrappurification. 20
Table 5.Determination ofamountofrGST-COE two different
culturetypes. 52
Table6.PhysicalpropertiesofrGST-COE. 54
Table 7. Detection of sequence of purified rGST-COE
byelectrosprayionization(ESI). 55
Fi
gur
es
Figure1.Schemeofthestudy. 2
Figure 2.Schematic representation ofthe PEDV genome based
ontheCV777strain. 4
Figure3.PEDV spikeprotein. 5
Figure4.Thechaperonenetworkoftheprokaryoticcytosol. 7
Figure5.Vectormapofchaperoneplasmids. 22
Figure6.SDS-PAGE patternsforrGST-COE (a)andrGST-S1D
(b)withoutchaperonesystem (BL21)andwitheachof
pTf16orpGro7orpG-KJE8. 23
Figure 7.Confirmation ofchaperone effecton the solubility for
rGST-COE (a) and rGST-S1D (b) expression by
SDS-PAGE gel. 25
Figure 8.Confirmation ofprotein expression by SDS-PAGE gel
Figure 9. SDS-PAGE gel analysis of optimization of
rGST-COE expression at 37℃ (a),28℃ (b),21℃
(c)and15℃ (d). 29
Figure 10.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I) fractions and relative amountofrGST-COE (a)
and rGST-S1D (b) expression at different IPTG
concentration(0.1,0.4,0.7and1.0mM). 31
Figure 11.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I) fractions and relative amountofrGST-COE (a)
and rGST-S1D (b)expression atdifferentInduction
time(OD6000.6,0.9,1.2and1.5). 33
Figure 12.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I) fractions and relative amountofrGST-COE (a)
and rGST-S1D (b) expression at different harvest
time(12,24,36and48h). 35
Figure 13.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I)fractions and relative amountofrGST-COE (a)
and rGST-S1D (b) expression with comparison of
optimized 0.1 mM IPTG with lactose(1,5,10mM)
asinducer. 37
Figure 14. Purification ofrGST-COE by glutathionesepharose
4B method. 38
Figure 15. Growth curve of rGST-COE in LB broth and
syntheticmedium. 40
Figure 16.Growth curve ofrGST-COE at30℃ and 37℃ in
syntheticmedium. 42
Figure 17.SDS-PAGE gelanalysisofoptimization ofInduction
OD6007∼8atgrowth temperature30℃ (a)and 37℃
(b)forrGST-COE expression. 44
Figure 18.SDS-PAGE gelanalysisofoptimization ofInduction
expression. 46
Figure19.Optimization ofInductionOD600between 7∼8(a)and
3∼4(b) atgrowthtemperature30℃ forrGST-COE
expression. 48
Figure 20.Optimization ofL-arabinose concentration between
1.0 mg/ml(a)and 2.0 mg/ml(b) for rGST-COE
expression. 50
Figure 21.Optimization ofL-arabinose concentration between
0.5mg/mland 1.0mg/mlforrGST-COE expression
atdifferentharvesttime 15,18,21 and 24 h after
induction. 51
Figure 22. Purification of rGST-COE by GSTrap column
method. 53
Figure23.AminoacidsequenceofrGST-COE protein. 55
Li
stofAbbr
evi
at
i
ons
AA:Aminoacids
CAT:Chloramphenicol-acetyl-transferase
DO:Dissolvedoxygen
E.coli:Escherichiacoli
ESI:Electrosprayionization GST:Glutathiones-transferase HRP:Horseradishperoxidase
IPTG:Isopropyl-1-thio-â-D-galactopyranoside
LB:Luriabroth
MW:Molecularweight
OD:Opticaldensity
ORFs:Openreadingframes
PA:Penicillinacrylase
PBS:Phosphate-bufferedsaline
PED:Porcineepidemicdiarrhea
rGST:Recombinantglutathiones-transferase
RT:Room temperature
SDS-PAGE:Sodium dodecylsulfate-polyacrylamidegel
electrophoresis
TBST:Tris-BufferedSalineandTween20
TF:Triggerfactor
I
.I
nt
r
oduct
i
on
Among many hosts used for the production of recombinant
proteins, Escherichia coli has been the most widely used prokaryotic hostbecause itis easy to culture,has a shortlife cycleandiseasily manipulatedgenetically duetoitswell-known genetics.E.coli is often used to produce protein vaccine to
minimize the costofproduction.However,there are two main
problems forprotein expression in E.coli.The firstoneislow solubility ofrecombinantproteinsforoverexpression.Anotheris littleordifficultexpression ofaforeign gene(Choi,J.H.etal.,
2006,Gopal,G.J.andKumar,A.,2013).
Toovercometheseobstacles,twostrategiesareoftenused.The first strategy is chaperone co-expression which assists the
folding of newly synthesized proteins to the native and
circumvents inclusion body formation,therefore leading to an
improvedsolubilityoftherecombinantprotein(Sahdev,S.etal.,
2008).Thesecondstrategy ischanging thecultureconditionsfor screeningthegrowthorexpressiontemperature,theconcentration
of the inducer, the induction time and harvest time, thus
optimizing mass-production ofprotein (Francis,D.M.and Page,
R.,2010).
Inchapter1,weconfirmedpTf16chaperoneco-expressioneffect
and optimized production of protein by changing the culture conditions, expression temperature, inducer concentration, inductiontimeandharvesttimeinflasklevel.
culture conditions, growth temperature, induction time and concentration ofL-arabinosein fermenterleveland then purified
proteinbyGSTrapcolumnmethod.
In this study,we demonstrated chaperone co-expression effect and obtained greatly increased mass-production and enhanced efficiency ofprotein purification changing the culture conditions from flaskleveltofermenterlevel(Figure1).
I
I
.Revi
ew ofLi
t
er
at
ur
e
1.Por
ci
neepi
demi
cdi
ar
r
heavi
r
us
1)CharacteristicsofthePEDV
Porcineepidemicdiarrhea(PED)wasfirstreportedinEuropein
1971(Goldstein,T.etal.,2013).Thediseasewascharacterized
by devastating enteritis,vomiting,watery diarrhea,dehydration,
and a high mortality rate among swine. Subsequently, the
causative agent of PED was identified as porcine epidemic
diarrhea virus (PEDV),which is a coronavirus (Hinshaw,V.et
al. , 1986) and contains an enveloped and single-stranded
positive-sense RNA genome.PEDV has been reported in many
othercountries,including Germany,France,Switzerland,Hungary,
Italy,China,South Korea,Thailand,Vietnam (Hinshaw,V.etal.
,1984)andtheUnitedStates.
PEDV has a considerable economic burden given that it is
highly infectious,resulting in significantmortality and morbidity in piglets. mortality and morbidity rates were lower for vaccinated herds than for nonvaccinated herds,which indicates
the emergence of a new PEDV field strain(s) for which the
current vaccine, based on the CV777 strain, was partially
protective(Song,D.andPark,B.,2012).
PEDV is an enveloped virus possessing an around 28 kb,
positive-sense,single-strandedRNA genomewitha5’capanda
The genome consists ofa 50 untranslated region (UTR),a 30
UTR and seven open reading frames (ORFs) that encode 4
structuralproteins [spike (S),envelope (E),membrane (M),and nucleocapsid(N)]andthreenon-structuralproteins(replicases1a
and 1b,and ORF3);these are arranged on the genome in the
order 50-replicase (1a/1b)–S-ORF3–E–M–N–30 (Figure 2)
(De Vries, A.A. et al. , 1997, Murphy, F.A. et al. , 1999,
Pensaert,M.andDeBouck,P.,1978).
Figure 2.Schematic representation ofthe PEDV genome based
ontheCV777strain (GenBankaccessionNo.AF353511)(Song,D.
2)PEDV spikeprotein
ThePEDV Spike(S)protein isatypeIglycoprotein composed
of1,383aminoacids(aa).TheS proteincanalsobedividedinto
S1domain (1–789 aa)and S2 domain (790–1,383aa)based on
itshomology with S proteinsofothercoronaviruses.Itcontains
a signalpeptide (1–18 aa),a transmembrane domain (1,334–
1,356 aa),a shortcytoplasmic domain and neutralising epitopes
including partialS1 and S2 domain (Figure3)(Chang,S.-H.et
al.,2002,Cruz,D.J.M.etal.,2008,Godet,M.etal.,1994,
Jackwood,M.W.etal.,2001,Sturman,L.S.and Holmes,K.V.,
1984, Sun, D. et al. , 2008).The PEDV S protein is a
glycoprotein peplomer (surface antigen)on the viralsurface.It plays an importantrole in regulating interactions with specific host cell receptor glycoproteins to mediate viral entry and stimulatesinduction ofneutralizing antibodiesin thenaturalhost
(Bosch,B.J.etal.,2003,Chang,S.-H.etal.,2002,Cruz,D.J.M.
etal.,2008,Godet,M.etal.,1994,Sun,D.etal.,2008).
Furthermore,itisassociatedwithgrowthadaptationinvitro,and
attenuationofvirulenceinvivo(Park,S.-J.etal.,2007,Sato,T.
etal.,2011).Therefore,theS glycoprotein would beaprincipal
targetforthedevelopmentofeffectivevaccinesagainstPEDV.
2.St
r
at
egi
est
oopt
i
mi
zemass-pr
oduct
i
on of
sol
ubl
epr
ot
ei
n expr
essi
on
1)Chaperoneco-expression
Chaperones assistthe folding ofnewly synthesized proteins to thenativestateandprovideaquality controlsystem thatrefolds misfolded and aggregated proteins.In the E.colicytosol,the folding of newly synthesized proteins is assisted by three systems composed of the ribosome-associated Trigger Factor
(TF)system,theDnaK/DnaJ/GrpE system andtheGroEL/GroES
system.TF system protects nascentchains from digestion by proteases and prevents misfolding by delaying folding until translationcompletes.DnaK/DnaJ/GrpE system stabilizesunfolded moleculesandpreventstheiraggregationuntilunfoldedmolecules fold properly. GroEL/GroES system transforms misfolded and aggregated protein into nativeprotein (Figure4)(Young,J.C.et
al.,2004).
The overproduction ofrecombinantproteins in hostcells often leads to their misfolding and aggregation.Folding problems of overproduced targetproteinscan becaused by limitationsin the chaperone capacity of the host cells.Chaperone co-expression strategy has been followed in many instances for inhibiting
inclusion body formation, thereby leading to an enhanced
Figure4.Thechaperonenetworkoftheprokaryoticcytosol
2)Modification ofthecultureconditions
(1)Theeffectoftemperature
Lowering the expression temperature routinely improves the solubility ofexpressed recombinantproteins (Kataeva,I.etal.,
2005,Shirano,Y.andShibata,D.,1990,Volontè,F.etal.,2008).
At lower temperatures,cell processes slow down,leading to reducedratesoftranscription,translation andcelldivision (Chou, C.P.,2007),while also leading to decreased protein aggregation
(Sahdev,S.etal.,2008).Furthermore,mostproteases are less
active at lower temperatures and therefore lowering the
expression temperature also results in a reduction in the degradation ofproteolytically sensitiveproteins(Pinsach,J.etal. ,2008).Expression atlow temperature conditions leads to the increased stability and correctfolding patterns (Shirano,Y.and
Shibata,D.,1990),which isbecauseofthefactthathydrophobic
interactionsdetermining inclusion body formation aretemperature
dependent(Sahdev,S.etal.,2008).The enhanced expression
and activity at lower growth temperatures has also been
associated with increased expression ofa numberofchaperones
in E.coli(Ferrer,M.etal.,2003).Ithas been reported that
heatshock proteases induced during over-expression are poorly
active at lower temperature conditions. Thus, growth at a
temperaturerange of15-23℃ leads to a significantreduction in degradationoftheexpressedprotein(Hunke,S.andBetton,J.M.,
(2)Theeffectofinducerconcentration
In addition to lowering the growth temperature,a reduction in transcription rate can also be achieved by lowering the inducer concentration.Decreasing the concentration of IPTG can also
enhances the production of soluble protein.For example,the
solubility of recombinant cyclomaltodextrinase (CDase) was shown tobehighly sensitivetotheconcentration oftheinducer.
When theprotein wasinducedusing 0.05mM IPTG,theprotein
wassolubleandactive.However,when theinducerconcentration
wasdoubledto0.1mM,theexpressedprotein wasinsolubleand
inactive (Turner,P.et al.,2005).Thus,although the most
commonIPTG concentrationsforproteininductionrangefrom 0.1 to1.0mM,adecreaseto even lowerlevelscan effectsolubility. As a result of metabolic burden (sometimes called metabolic load),the high concentrations ofinducerdo notnecessarily lead
tomaximalexpression ofatargetprotein (Glick,B.R.,1995)and
optimalinducerconcentration on the efficiency ofthe induction
(3)Theeffectofinduction time
We generally know that foreign protein expression causes a metabolicburden on thecellwhich can resultin reducedgrowth rates, cell yields, product expression and plasmid stability
(Bentley,W.E.etal.,1991,Bentley,W.E.etal.,1990,Glick,
B.R.,1995).WhilethelevelofIPTG inducerused can bevaried
toadjusttheextentofthemetabolicburden imposedon thecell,
themaximum productofforeign protein forexpression willalso
depend on thepointin thegrowth cycleatwhich expression is
induced.Forstrains whose growth and viability are drastically decreased following induction,induction in late-log orstationary phaseprovideshighcelldensitiesforincreasedproductformation. However,asshownforchloramphenicol-acetyl-transferase(CAT)
expression underthe controlofthetac promoter(Bentley,W.E.
etal.,1991),low growth ratesandproteaseactivity broughton
by depleted nutrientlevels in the stationary phase can decrease theyieldofforeignprotein.
In this case,optimalinduction in the mid-log phase provided sufficientlevelsofCAT proteinwithinthecellwhileachieving a
high celldensity to produce the maximalyield.When product
expression does not significantly influence cellgrowth,foreign proteinyieldismaximizedby inducing expression throughoutthe
entiregrowth phase.Thiswasshown forproduction ofsecreted
penicillin acrylase (PA)in E.coliunderthe controlofthe lac
promoter (Ramirez,O. et al., 1994),where early-log phase
induction was optimalbecause PA transport to the periplasm ratherthantheenergylevelavailabletothecellwasthelimiting factor.
I
I
I
.Mat
er
i
al
sandMet
hods
1.Opt
i
mi
zat
i
on off
l
askcul
t
ur
econdi
t
i
ons
1)Strainsandmedia
pGEX6p-1 expressing rGST-COE and pGEX6p-1 expressing
rGST-S1D withpTf16orpGro7orpG-KJE8werereceivedfrom
our lab member (Table 1).pTf16,pGro7 and pG-KJE8 were
purchasedfrom Takarabio.
The culture medium used for allexperiments was LB broth
containing 10 g/ltryptone,5 g/lyeastextractand 10 g/lNaCl
(pH 7.0).
Table1. Plasmidsusedinthisstudy.
Protein Cloning region
(aminoacidNo.) Vector
Chaperone
plasmid/Strain
rGST-COE 499-636 pGEX-6p-1 pTf16/BL21
rGST-COE 499-636 pGEX-6p-1 pGro7/BL21
rGST-COE 499-636 pGEX-6p-1 pG-KJE8/BL21
rGST-S1D 637-789 pGEX-6p-1 pTf16/BL21
rGST-S1D 637-789 pGEX-6p-1 pGro7/BL21
2)Cultureconditions
Shakeflaskexperimentswereperformedinshaking incubatorat
230rpm and37℃,using25mlofLB brothcontaining0.5mg/ml
L-arabinose,100 µg/mlampicillin and 20 µg/mlchloramphenicol
(Table2)in 100mlbaffle-equiped shakeflasks.A 5 mlculture
tubewasinoculatedwiththebacterialglycerolstocksolutionand
grownovernightontheLB brothcontaining 100µg/mlampicillin
andthenusedasseedforeachexperiment.Thebiomassincrease
was checked by measuring the absorbance at600 nm with a
spectrophotometer.WhentheOD600oftheculturesolutionreaches
induction time,leaveat4℃ for30min.Then cellswasinduced
by specified IPTG atspecified expression temperature overthe specifiedinductiontimeandharvesttime.
Table2. Chaperoneteam codedoneachplasmidandinducer.
Plasmid Chaperone Promoter Inducer(final
concentration) Antibiotic
pTf16 Trigger
factor AraB
L-Arabinose (0.5mg/ml)
Chloramphenicol (20μg/ml)
pGro7 GroEL/GroES AraB L-Arabinose (0.5mg/ml) Chloramphenicol (20μg/ml) pG-KJE8 DnaK/DnaJ/ GrpE, GroEL/GroES AraB Pzt-1
L-Arabinose (0.5mg/ml), Tetracycline
(5ng/ml)
Chloramphenicol (20μg/ml)
3)Chaperoneco-expression screening
To express rGST-COE or rGST-S1D, E.coli system with
pTf16orpGro7orpG-KJE8orwithoutchaperonewerecultured
in flasks in shaking incubator in parallel, and each culture
system wasinduced with 0.1mM IPTG when theOD600reached
0.6.Theflaskswereincubatedfor24hat15℃
4)Sonication
Biomasswasharvestedattheendoftheproduction processby
centrifugation at 5,000 rpm and the wet bacterialpellets was
re-suspended in PBS (140 mM NaCl,2.7 mM KCl,10 mM
Na2HPO4,1.8 mM KH2PO4,pH 7.3).Samples were kepton ice
and disrupted by sonication using a program consisting of45
cycles (2”on,5”off,amp 20 %).Afterdisruption ofthe cells,
sampleswerecentrifugedat14,000rpm for15minataconstant
temperature of4℃.The pellets and supernatantwere stored at
–20℃.
5)SDS-PAGE
Aftersonication,the supernatantand the pellets (re-suspended
in 50 mM Tris-HCl,pH 12.5)were separately mixed with 5X
SDS-PAGE loading bufferand incubated at100℃ for5minutes.
After incubation, the samples were loaded into 10 % (w/v)
Tris-Glycine SDS-PAGE pre-cast gel (Komabiotech, Korea).
Electrophoresiswasconductedat50V for30minutesand130V
thegelswereanalyzedbyimagelabsoftware(BioRad,USA).
6)Western blot
In western blot assay, separated supernatant proteins using
SDS-PAGE were transferred to a nitrocellulose membrane
(Whatman,USA).The transferred membrane was blocked with
TBST containing 5 % skim milk (BD Biosciences)for1 h at
room temperature and probed with mouse anti-GST monoclonal
antibody atRT for1h.Thesignalwasdetected by using goat
anti-mouse secondary antibody conjugated to horseradish
peroxidase (HRP,Millipore)for1 h with Amersham ECL prime
westernblottingdetectionreagentassubstrate(GE Healthcare).
7)Optimization ofexpression temperature
To express each protein,each culture was induced atdifferent
expressiontemperature15,21,28and37℃ andbyusing 0.1mM
IPTG atOD6000.6toeach oftheculturemedia.Theflaskswere
incubatedfor24h(15℃),16h(21℃),8h(28℃)and4h(37℃).
8)Optimization ofIPTG concentration
Fourflasks in shaking incubatorwere performed in parallelto express each protein.Allcultures were grown untilthe OD600 reached 0.6 and each culturewas induced with 0.1,0.4,0.7 and
1.0mM IPTG concentration.Theflaskswereincubated for24h
9)Optimization ofinduction time
Fourflasks in shaking incubatorwere performed in parallelto expresseach protein.Allculturesweregrown untilthedifferent
OD600 reached 0.6,0.9,1.2 and 1.5,and then each culture was
inducedwith0.1mM IPTG for24hat15℃.
10)Optimization ofharvesttime
Fourflasks in shaking incubatorwere performed in parallelto express each protein.Allcultures were grown untilthe OD600
reached 0.6 and each culture was induced with 0.1 mM IPTG.
Theflaskswereincubated for12,24,36and 48h harvesttime
at15℃.
11)Comparison ofIPTG withlactoseasinducer
Fourflasks in shaking incubatorwere performed in parallelto express each protein.Allcultures were grown untilthe OD600
reached 0.6.one culture was induced with 0.1 mM IPTG and
others were induced at 1, 5 and 10 mM lactose for 12 h
(rGST-S1D)and24h(rGST-COE)at15℃.
12)Purification ofrGST-COE from solublefraction
The biomass harvested at the end of the cultivations was
disruptedby sonication in BufferA atpH 7.3(usedasthelysis
and 1.8 mM KH2PO4)and centrifuged at14,000 rpm for15 min to obtain the crude soluble protein (Table 3,4).Crude soluble protein wasapplied to glutathionesepharose4B (GE Healthcare) equilibrated with BufferA (equilibration buffer).Then BufferA
(used as washing buffer)was applied to the column to wash
unboundedmaterial.BufferB (usedaselutionbuffercomposedof
50mM Tris-HCland10mM glutathioneatpH 8.0)wasapplied
2.Devel
opmentoff
er
ment
at
i
on pr
oduct
i
on
pr
ocessand ant
i
gen pur
i
f
i
cat
i
on
1)Strain andmedia
pGEX6p-1expressing rGST-COE withpTf16wasreceivedfrom
ourlabmember.
The culture media used for experiments were LB broth and
syntheticmedium.
FirstLB broth consisted of10g/ltryptone,5g/lyeastextract
and10g/lNaCl,pH 7.0.
Second synthetic medium consisted of3.5 g/lKH2PO4,5.0 g/l
K2HPO4, 3.5 g/l (NH4)2HPO4, 0.5 g/l MgSO4⋅7H2O, 30 g/l
glucose,5.0g/lyeastextract,0.5ml/lantifoam and1.0ml/ltrace metals,pH 7.0.Trace metals formulation consisted of 1.6 g/l
FeCl3,0.2g/lCoCl2⋅6H2O,0.1g/lCuCl2,0.2g/lZnCl2⋅4H2O,0.2
g/lNaMoO4,0.05g/lH3BO4,and10mlHCl,to1,000ml.
2)Cultureconditions
E. coli colonies were picked up from LB agar plate and
transferred to the culture medium forovernightincubation at3 7℃, 230 rpm. The cultured cells were transferred to fresh medium in flask and incubated for 3 hr.finally,the fresh cell
culture was inoculated to batch fermenter with a volume
correspondingto5% ofthefermentercultivationvolume.
Experimentwascarried outin a 1.5L fermenter.E.coliBL21
brothand1L syntheticmedium containing specifiedL-arabinose,
100 µg/mlampicillin and 20 µg/mlchloramphenicol.When the
OD600oftheculturesolution reachesspecifiedinduction time,the
culturewascooled to15℃ for1h.Then thecellswereinduced
with 0.1mM IPTG atexpressiontemperature15℃.Threeprobes
composed of pH probe, thermal probe and dO2 probe were
employed.Dissolved O2concentration at30% ofsaturation was
maintainedbychanging theagitationspeed(between400and600
rpm)and enriching airwith oxygen.Thetotalairflow rate was
rangedbetween1and1.5VVM.ThepH wascontrolledat7.0±
0.2 by adding 1 M NaOH (base)and 1 M HCl(acid).Culture
samples (6.25 ml) were regularly withdrawn to check the cell
growth by measuring the absorbance at600 nm.The samples
were then centrifuged at14,000 rpm and 4°C for15 min.The
supernatantswereseparatedandfrozenat -20°C.
3)Optimization ofgrowth temperature
FourE.colisystemswereculturedin fermenters.Twocultures
weregrown untiltheOD600reached 3∼4at30℃ and 37℃.The
others were grown untilthe OD600 reached 7∼8 at30℃ and 3
7℃.Theneachculturewasinducedwith 0.1mM IPTG for24h
at15℃.
4)Optimization ofinduction time
Two fermenters were performed in parallel.Allcultures were
grown untiltheOD600reached3∼4and7∼8at30℃.Then each
5)Optimization ofL-arabinoseconcentration
Three fermenters were performed in parallel.Allcultures were
grown untiltheOD600reached 3∼4at30℃ with 0.5,1.0and 2.0
mg/mlL-arabinose.Theneachculturewasinducedwith0.1mM
IPTG for24hat15℃.
6)Purification ofrGST-COE from solublefraction
The biomass harvested at the end of the cultivations was
disruptedby sonication in BufferA atpH 7.3(usedasthelysis
buffercomposedof140mM NaCl,2.7mM KCl,10mM Na2HPO4
and 1.8 mM KH2PO4)and centrifuged at14,000 rpm for15 min
to obtain the crude soluble protein (Table 3,4).Crude soluble
protein wasapplied toGSTrap column (175mm dimensions,GE
Healthcare) equilibrated with Buffer A (equilibration buffer).
Then Buffer A (used as washing buffer) was applied to the
column to wash unbounded material.BufferB (used as elution
buffercomposed of50 mM Tris-HCland 10 mM glutathioneat
pH 8.0) was applied to the column to obtain purified protein
Table3.Buffersforcolumnscreening.
Buffers Description
BufferA
(Lysis/Equilibration/
Washing) 140mM NaCl,2.7mM KCl, 10mM Na2HPO4and1.8mM KH2PO4 atpH 7.3 BufferB (Elution) 50mM Tris-HCland 10mM glutathioneatpH 8.0
Table4.ProcedureofGSTrappurification.
Stepsand buffers Fractionsandamount Flow rate
Columnequilibration
(BufferA) Columnvolumex 5 5ml/min
Sampleflow through Samplesupernatants
volume 1ml/min
Washing(BufferA) Washing fraction1,2
(Columnvolumex5) 5ml/min
Elution(BufferB) Elutionfraction1,2,3
I
V.Resul
t
sandDi
scussi
on
Chapt
er1.Opt
i
mi
zat
i
on off
l
ask cul
t
ur
e
condi
t
i
ons
1.Chaper
oneco-expr
essi
on
1)Chaperoneco-expression screening
We compared the solubility ofPED antigen with three other
types ofchaperon system obtained from Takara bio (Figure 5):
pTf16(Triggerfactorsystem),pGro7(GroEL/GroES system)and
pGKJE8(DnaK/DnaJ/GrpE andGroEL/GroES system).
In ordertodeterminetheoptimalchaperonesystem,rGST-COE
and rGST-S1D were produced with differentchaperone systems
(pTf16,pGro7and pG-KJE8).Allculturesweregrown untilthe
OD600 reached 0.6 and each culture was induced with 0.1 mM
IPTG concentration.Theflaskswereincubated for24h at15℃.
By SDS-PAGE,theexpressionofthechaperonesystemsandthe
increaseoftherGST-COE andtherGST-S1D proteinforsoluble
form wereconfirmedforthesamplesfrom thecellswitheachof
pTf16orpGro7orpG-KJE8plasmids(Figure6).
The co-expression oftrigger factor in pTf16 plasmid showed greater solubility of PED antigen than other combinations of
chaperones. These results suggested that a combination of
chaperone in pTf16 plasmid may be the mostefficientway to
Figure 5.Vectormap ofchaperoneplasmids.(a)ThepG-KJE8
isfortheproduction ofdnaJ,dnaK,grpE,groEL andgroES.(b)
ThepGRO7isfortheproduction ofgroEL andgroES.(c)pTf16
is for the production of trigger factor. Arrows indicate the
directions of the genes (araB: araB promoter, araC: araC
repressor,dnaK:DnaK gene,dnaJ:DnaJgene,grpE:GrpE gene,
Figure 6. SDS-PAGE patterns for rGST-COE (a) and
rGST-S1D (b)withoutchaperone system (BL21)and with each
ofpTf16 orpGro7 orpG-KJE8.Arrow indicates PED antigen.
MolecularsizeofrGST-COE is41.4kDaandrGST-S1D is42.8
2)Confirmation ofchaperoneeffect
The SDS–PAGE showed the expression oftrigger factor (56
kDa)inducedby 0.5mg/mlL-arabinose(Figure7.dottedarrow)
andtheexpressionof rGST-COE andrGST-S1D inducedby0.1
mM IPTG (Figure7.solidarrow).Both PED antigenandtrigger
factorwasnotproducedwithoutL-arabinoseandIPTG induction.
Trigger factor was only produced with L-arabinose.Insoluble
form PED antigen was produced with IPTG in the absence of
L-arabinose.Soluble form PED antigen was produced in the
presence ofchaperone (trigger factor).Chaperone co-expression increasedrecoveryofproteinsinsolublefraction.Asaresult,we confirmed thatco-expression ofpTf16improved thesolubility of
Figure 7.Confirmation ofchaperone effecton the solubility for
rGST-COE (a) and rGST-S1D (b) expression by SDS-PAGE
gel.M,markersin kDa;S,solublefraction;I,insolublefraction.
Lanes: –/–, no induction; A/–, induction with 0.5 mg/ml
L-arabinosealone;–/I,induction with 0.1mM IPTG alone;A/I,
induction with 0.5 mg/mlL-arabinose and 0.1 mM IPTG.The
solid arrow indicates rGST-COE (a) and rGST-S1D (b)
2.Conf
i
r
mat
i
on ofpr
ot
ei
n expr
essi
on by
west
er
n bl
ot
After determining the optimalchaperone system,western blot was conducted in orderto validate thatproduced proteins were
rGST-COE and rGST-S1D.The expression ofPED antigen,as
determined by western blot using anti-GST antibody, was
confirmed in soluble fraction when trigger factor was
Figure 8.Confirmation ofprotein expression by SDS-PAGE gel
(a)andwesternblot(b)usinganti-GST antibody.M,markersin
kDa;S,soluble fraction;I,insoluble fraction.The solid arrow
indicates rGST-S1D and the dotted arrow indicates rGST-COE
3.Modi
f
i
cat
i
on oft
hecul
t
ur
econdi
t
i
ons
1)Optimization ofexpression temperature
In order to determine the optimal expression temperature, rGST-COE wasproducedatdifferentexpressiontemperature(37,
28,21 and 15℃),while thetriggerfactorwas induced with 0.5
mg/mlL-arabinose.SDS-PAGE showed thatsolublerGST-COE
expression was very low at 37℃ and very high at 15℃
(Figure9).Theseresultsshowedthatthelowerthetemperature,
thehigherthePED antigenexpressionbecauselowertemperature
reduced rates of transcription, translation and cell division, leading todecreasedproteinaggregation(Francis,D.M.andPage,
R.,2010).As a result,15℃ is the most suitable expression
Figure9.SDS-PAGE gelanalysisofoptimizationofrGST-COE
expression at 37℃ (a),28℃ (b),21℃ (c) and 15℃ (d).M,
markers in kDa;S,soluble fraction;I,insoluble fraction.The solidarrow indicatesrGST-COE expression.
2)Optimization ofIPTG concentration
Afterdetermining theoptimalchaperonesystem and expression temperature, the effect of the IPTG concentration on the efficiency ofthe induction was examined by using 0.1,0.4,0.7
and 1.0 mM IPTG concentrations. The relative amount of
rGST-COE and rGST-S1D and corresponding proteins in the
soluble and insoluble fractions at different IPTG concentration
were shown in SDS-PAGE gel(Figure 10).According to the
results,solubleform expression ofPED antigen wasthehighest with 0.1 mM IPTG concentration with the lowestproduction of
Insolubleform.Insolubleform expressionofPED antigenwasthe
highestat1.0mM IPTG concentrationwiththelowestproduction
of solubleform.Itindicatedthatthehighertheconcentration of
IPTG,the higher the production of insoluble form with low
production of soluble protein.As a result,the optimal IPTG
concentration was 0.1 mM for the soluble expression of PED
Figure 10.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I) fractions and relative amount of rGST-COE (a) and
rGST-S1D (b) expression atdifferentIPTG concentration (0.1,
0.4,0.7and 1.0mM).M,markersin kDa;S,solublefraction;I,
3)Optimization ofinduction time
After determining the optimal chaperone system, expression temperatureand IPTG concentration,theeffectofinduction time ontheefficiency oftheinductionwasexaminedatOD6000.6,0.9,
1.2 and 1.5.The relative amountofrGST-COE and rGST-S1D
and corresponding proteins in thesolubleand insolublefractions
at different induction time were shown in SDS-PAGE gel
(Figure 11).Results showed thatthe higherinduction time,the higherthe production ofinsoluble form with low production of
solubleproteinexceptrGST-COE atOD6001.5.Solubleexpression
ofPED antigen was atits bestatinduction OD6000.6 forboth
rGST-COE and rGST-S1D.As a result,atinduction OD600 0.6,
the cells were healthy and metabolically active and thus the
Figure 11.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I) fractions and relative amount of rGST-COE (a) and
rGST-S1D (b)expression atdifferentInduction time (OD6000.6,
0.9,1.2 and 1.5).M,markers in kDa; S,soluble fraction;I,
4)Optimization ofharvesttime
After determining the optimal chaperone system, expression temperature,IPTG concentration and induction time,theeffectof harvesttimeon theefficiency oftheinduction wasexaminedfor
12,24,36 and 48 h.The relative amount of rGST-COE and
rGST-S1D and corresponding proteins in the soluble and
insoluble fractions at different harvest time were shown in
SDS-PAGE gel (Figure 12).Results showed that the higher
harvesttime,thehighertheproduction ofinsolubleform andthe
lowerharvesttime,thehighertheproduction ofsolubleform in
Figure 12 (b).With the increasing harvesttime,itseems that the toxicity ofexpressed proteins kills the cells converting the solubleproteinsintoinsolubleform.Asaresult,theproductionof
solublePED antigenwashighestfor24h(Figure12(a))and12
h (Figure 12 (b))harvesttime forrGST-COE and rGST-S1D
Figure 12.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I) fractions and relative amount of rGST-COE (a) and
rGST-S1D (b)expression fordifferentharvesttime (12,24,36
and 48 h).M,markers in kDa;S,soluble fraction;I,insoluble
5)Comparison ofIPTG with lactoseasinducer
After determining the optimal chaperone system, expression temperature,IPTG concentration,inductiontimeandharvesttime,
wecompared ofoptimized 0.1 mM IPTG with lactose(1,5 and
10mM)asinduceronefficiencyoftheinduction.
The relative amount of rGST-COE and rGST-S1D and
corresponding proteinsin thesolubleand insolublefractionswith
comparison ofIPTG with lactosewereshown in SDS-PAGE gel
(Figure 13).Results showed thatthe production ofsolubleform
with 0.1 mM IPTG was higherthan the production ofsoluble
form at1,5 and 10 mM lactose.When compared to 0.1 mM
IPTG,1,5and 10mM lactoseinduced lowerproduction ofPED
antigen in insolubleform.However,when compared to 1,5 and
10mM lactose,0.1 mM IPTG induced thehighestproduction of
PED antigen in soluble form.Therefore,0.1 mM IPTG is more
efficientinducerthan lactose(1,5and 10mM)forsolubleform expression.
Figure 13.SDS-PAGE gelanalysisofsoluble(S)and insoluble
(I) fractions and relative amount of rGST-COE (a) and
rGST-S1D (b)expression with comparison ofoptimized 0.1 mM
IPTG withlactose(1,5,10mM)asinducer.M,markersinkDa;
4. Pur
i
f
i
cat
i
on of r
GST-COE f
r
om
sol
ubl
e
f
r
act
i
on
1)Glutathionesepharose4B method
The cells were lysed using sonication and the soluble fraction obtained by centrifugation was loaded on Glutathione sepharose
4B equilibrated with bufferA (140 mM NaCl,2.7 mM KCl,10
mM Na2HPO4and 1.8mM KH2PO4atpH 7.3).Thecolumn was
washedsuccessivelywithbufferA. TherGST-COE proteinwas
eluted using bufferB (50mM Tris-HCland 10mM glutathione
atpH 8.0)andfractionscontaining therGST-COE werecollected
(Figure 14).Concentration of purified soluble protein at flask
levelwas5mg/l.
Figure 14.Purification ofrGST-COE by glutathione sepharose
4B method.Arrow indicates rGST-COE expression.Lane 1 :
solublefraction beforeapplying tocolumn,Lane2:sampleflow
Chapt
er
2. Devel
opment of f
er
ment
at
i
on
pr
oduct
i
on pr
ocessandant
i
gen pur
i
f
i
cat
i
on
1.Moni
t
or
i
ng gr
owt
h cur
ve
1)Comparison ofculturemedium forfermentation
(1)Between LB broth andsyntheticmedium
The growth behaviourofrGST-COE was investigated at37℃
in LB broth and synthetic medium withoutcontrolofpH and
DO.A lag phage of around two hours was observed in the
growth ofthecellsin both media.Theexponentialphaseofcell
growth wascompleted with 5and 7 hoursofinoculation in LB
broth and synthetic medium respectively,after which the cells
entered theirstationary phase.Thegrowth curvewas shown in
Figure 15.Glucose in synthetic medium is main carbon and
energy source.When cellsconsumedglucose,cellsachievedhigh celldensity.Therefore,maximum OD600 ofsynthetic medium is
twotimeshigherthanthatofLB broth.Asaresult,weselected
Figure15.GrowthcurveofrGST-COE inLB brothand
2)Comparison ofgrowthtemperatureforfermentation
(1)Between growth temperature30℃ and37℃
Afterdetermining syntheticmedium,weinvestigatedthegrowth
behaviourofrGST-COE at30℃ and 37℃ in synthetic medium.
We maintained the DO levelhigher than 30% with increasing
rpm andmaintainedpH 7withcontrolling1M NaOH (base)and
1M HCl(acid).At growth temperature 30℃,a lag phage of
around 3 hours was observed in the growth ofthe cells.The
exponentialphaseofcellgrowthwascompletedwith 13.5hours,
afterwhich the cells entered theirstationary phase.Atgrowth
temperature37℃,alagphageofaround2hourswasobservedin
the growth ofthe cells.The exponentialphase ofcellgrowth
wascompleted with 7 hours,afterwhich the cellsentered their
stationary phase.The growth curve was shown in Figure 16.
When pH and DO levelwere not controlled,maximum OD600
value was 10.1 at37℃.However,afterpH and DO levelwere
controlled,maximum OD600valuewasenhancedfrom 10.1to15.6
at37℃.Also,we showed thatcells grow much fasterat37℃
than30℃.AlthoughtheOD600valuereachedmaximum quicklyat
37℃ than 30℃,themaximum OD600valuewassimilaratboth
temperatures.ThearrowsonthegrowthcurveindicatetheOD600
valuesofthetwo phasesofcellgrowth.Induction ofcellswere
conducted attwo stages ofgrowth.Firstsolid arrow indicates
early-log phaseatinduction OD6003∼4andsecond dottedarrow
indicatesmid-log phaseatinductionOD6007∼8at30℃ and37℃
Figure 16.Growth curve ofrGST-COE at30℃ and 37℃ in
synthetic medium. Solid arrow indicates early-log phase at
induction OD6003∼4 and second dotted arrow indicates mid-log
2.Opt
i
mi
zat
i
on ofbat
ch t
ypeexpr
essi
on
1) Optimization of growth temperature 30℃ and 37℃ at
induction OD6007∼8
Inordertodeterminetheoptimalgrowthtemperature,cellswere
grown untiltheinduction OD6007∼8atgrowth temperature30℃
and 37℃ with 0.5 mg/ml L-arabinose. Then the cells were
inducedwith0.1mM IPTG for3,6,9,12,15,18,21and24hat
expression temperature15℃.SDS-PAGE showed thatthehigher
the harvesttime,the higherthe production ofsoluble form for rGST-COE expression (Figure 17).With the increasing harvest
time,it seemed that cells produced higher amount of soluble
proteins.Also SDS-PAGE showed thattheproduction oftrigger
factorproducedat30℃ washigherthantheproductionoftrigger
factor produced at 37℃. It appeared that there was a little
expression at30℃,buttherewasnoornegligibleexpression at
37℃.Theseresultssuggested thatthe growth temperature30℃
isbetterthan 37℃ atinduction OD6007∼8fortheexpression of
Figure 17.SDS-PAGE gelanalysisofoptimization ofInduction
OD600 7∼8 at growth temperature 30℃ (a) and 37℃ (b) for
rGST-COE expression.Arrow indicates rGST-COE expression.
Lane1:justbeforeinduction,Lane2-9:harvesttimeof3,6,9,
2) Optimization of growth temperature 30℃ and 37℃ at
induction OD6003∼4
Afterexamining growth temperature30℃ and 37℃ atinduction
OD6007∼8,we tested the growth temperature30℃ and 37℃ at
induction OD6003∼4.Cellsweregrown untiltheinduction OD600
3∼4 at growth temperature 30℃ and 37℃ with 0.5 mg/ml
L-arabinose.Then cellswereinduced with 0.1 mM IPTG for6,
9,12,15,18,21,24 and 40 h atexpression temperature 15℃.
Similartoaboveresults,SDS-PAGE showed thathigherharvest
time,the higherthe production ofsoluble form forrGST-COE
expression (Figure 18). With the increasing harvest time, it appeared that cells accumulate soluble protein. Production of
trigger factor produced at30℃ was higher than production of
triggerfactorproduced at37℃.SDS-PAGE showed thatwhen
the induction was done early,the protein expression increased
with timeat30℃,buttherewasalittleprotein expression at3
7℃.These results suggested thatthe growth temperature 30℃
was betterthan 37℃ atinduction OD600 3∼4 forexpression of
Figure 18.SDS-PAGE gelanalysisofoptimization ofInduction
OD600 3∼4 atgrowth temperature 30℃ (a)and 37℃ (b) for
rGST-COE expression.Arrow indicates rGST-COE expression.
Lane1 :justbeforeinduction,Lane2-9 :harvesttimeof6,9,
3)Optimization ofinduction OD6003∼4anad 7∼8atgrowth
temperature30℃
Weselectedoptimalgrowth temperature30℃ atinduction OD600
3∼4and 7∼8.In ordertodetermineoptimalinduction OD600,we
compared induction OD600 3∼4 and 7∼8 atgrowth temperature
30℃.Cells were grown untilthe induction OD6003∼4 and 7∼8
atgrowth temperature 30℃ with 0.5 mg/mlL-arabinose.Then
cellswereinduced with 0.1mM IPTG for3,6,9,12,15,18,21
and24h(Figure19(a))andfor6,9,12,15,18,21,24and40h
(Figure19(b))atexpression temperature15℃.Similartoabove
results,SDS-PAGE showed that the higher harvest time,the
highertheproduction ofsolubleform forrGST-COE expression
(Figure 19).With the increasing harvesttime,itappeared that
cells accumulate soluble protein.Production of rGST-COE and
triggerfactoratinductionOD6003∼4washigherthanproduction
ofrGST-COE and triggerfactoratinduction OD6007∼8 atthe
same growth temperature 30℃. In conclusion, these results
suggestedthattheinductionOD6003∼4wasbetterthaninduction
OD600 7∼8 for the expression of proteins at the growth
Figure19.Optimization ofInductionOD600between 7∼8(a)and
3∼4 (b)atgrowth temperature30℃ forrGST-COE expression.
Arrow indicatesrGST-COE.Lane1:justbeforeinduction,Lane
2-9 :harvesttime of3,6,9,12,15,18,21 and 24 h after
induction (a).Lane1:justbeforeinduction,Lane2-9:harvest
timeof6,9,12,15,18,21,24and 40 h afterinduction (b).M,
4)Optimization ofL-arabinoseconcentration
Afterdetermining growth temperature30℃ and induction OD600 3∼4,theeffectoftheL-arabinoseconcentrationontheefficiency
of the induction was examined by using 1.0 mg/mland 2.0
mg/mlL-arabinose concentrations.Cells were grown untilthe
induction OD6003∼4atgrowth temperature30℃ with 1.0mg/ml
and 2.0 mg/mlL-arabinose.Then cells were induced with 0.1
mM IPTG for 3,6,9,12,15,18,21 and 24 h atexpression
temperature 15℃.Similarto above results,SDS-PAGE showed
that theg/ml and 1.0 mg/ml L-arabinose concentration were
shown in SDS-P higherharvesttime,thehighertheproduction
of soluble form for rGST-COE expression. Also with the
increasing harvesttime,itappeared thatcellsaccumulatesoluble
protein. SDS-PAGE showed that production of rGST-COE
expression was higher at1 mg/mlthan 2 mg/mlL-arabinose
concentration (Figure 20). Then we compared the protein
expression between 0.5 mg/ml and 1 mg/ml L-arabinose at
growth temperature 30℃ and induction OD600 3∼4.The relative
amountofrGST-COE and corresponding proteins in the soluble
fractionsat0.5mAGE gel(Figure21).Thehigherconcentration
ofL-arabinose enhanced the higherexpression oftriggerfactor
butlowered theexpression ofrGST-COE.Theconsequencewas
mightbe due to consumption ofL-arabinose as inducerforthe
higher production of trigger factor that lowered the energy
source for the expression of rGST-COE.Finally,the growth
temperature 30℃,induction OD600 3∼4,0.5 mg/mlL-arabinose
were the mostfavorable conditions for the highestrGST-COE
Figure 20.Optimization ofL-arabinose concentration between
1.0 mg/ml(a) and 2.0 mg/ml(b) for rGST-COE expression.
Arrow indicatesrGST-COE.Lane1:justbeforeinduction,Lane
2-9 :harvesttime of3,6,9,12,15,18,21 and 24 h after
Figure21.OptimizationofL-arabinose concentrationbetween
0.5mg/mland1.0mg/mlforrGST-COE expressionatdifferent
harvesttimeof15,18,21and24hafterinduction.M,markers
3. Pur
i
f
i
cat
i
on of r
GST-COE f
r
om
sol
ubl
e
f
r
act
i
on
1)GSTrapcolumn method
The cells were lysed using sonication and the soluble fraction
obtained by centrifugation was loaded on GSTrap column
equilibrated with bufferA (140 mM NaCl,2.7mM KCl,10mM
Na2HPO4 and 1.8 mM KH2PO4 at pH 7.3).The column was
washedsuccessivelywithbufferA. TherGST-COE proteinwas
eluted using bufferB (50mM Tris-HCland 10mM glutathione
atpH 8.0)andfractionscontaining therGST-COE werecollected
(Figure 22). Concentration of purified soluble protein at fermentationlevelwas53.8mg/l.Finally,purifiedsolubleyprotein
yield was enhanced by 10 times from flask level(5 mg/l) to
fermentationlevel(53.8mg/l)(Table5).
Table5.DeterminationofamountofrGST-COE twodifferent
culturetypes.
Culturetype Finalcelldensity (OD) Total protein (g/l) Purified protein (mg/l) Purity (%) Flask 3 1.5 5 50 Fermenter 10 5 53.8 95
Figure 22. Purification of rGST-COE by GSTrap column
method.Arrow indicatesrGST-COE expression.Lane1:soluble
fraction before applying to column, Lane 2-3 : sample flow
4. El
ect
r
ospr
ay i
oni
zat
i
on assay of pur
i
f
i
ed
r
GST-COE pr
ot
ei
n f
r
om sol
ubl
ef
r
act
i
on
Electrospray ionization (ESI)resultshowed thatsome peptides of purified rGST-COE protein were detected.The amino acid sequence ofthe protein thatwe produced and purified matched
with the sequence ofrGST-COE thatwe had designed (Figure
23).This resultconfirmed thatthe purified protein is identical
withrGST-COE (Table6,7).
Table6.PhysicalpropertiesofrGST-COE.
Description Aminoacids MW [kDa] pI
Table 7. Detection of sequence of purified rGST-COE by electrosprayionization(ESI).
Absorbance Sequence
High MSPILGYWK
High LLLEYLEEK
High LTQSMAIIR
High IAYSKDFETLK
High IEAIPQIDKYLK
High KRIEAIPQIDK
High RIEAIPQIDK
High IEAIPQIDK
High GELITGTPKPLEGITD
V.Concl
usi
on
Porcine epidemic diarrhea (PED) is a contagious disease and
causessignificanteconomiclossesinmostoftheswinefarmsin
korea.In orderto develop efficientproductivity ofvaccines,we screenedchaperoneco-expressionsystem,expressiontemperature, induction time,harvesttime and concentration ofinducers such as IPTG (isopropyl-1-thio-â-D-galactopyranoside)and lactosein flaskcultureconditions.
Chaperones assistthe folding ofnewly synthesized proteins to thenativestateandprovideaquality controlsystem thatrefolds misfolded and aggregated proteins. In chapter 1, chaperone co-expression increased recovery of PED antigen in soluble
fraction.pTf16wasthemostsuitablechaperonesystem forPED
antigen expression.While lower temperature enhances the PED antigen expression, optimal expression temperature was 15℃.
Then western blotresultconfirmedtheexpressionofrGST-COE
and rGST-S1D.The higherthe IPTG concentration,the protein
expression rate also seems to be higherwith the formation of
higherinclusion bodies.Thus,0.1 mM IPTG concentration was
selected for the expression ofallthe proteins.IPTG induction with increasing OD600decreasedtheprotein expression.Thecells
were healthy and metabolically active and thus enhances the
maximum production ofsolubleform atinduction OD6000.6.The
higher the culture time,the higher the production ofinsoluble
form exceptrGST-COE.Theproduction ofsoluble PED antigen
washighestat24h and 12 h harvesttimeforrGST-COE and
rGST-S1D respectively.Theexpression with lactoseislowerin
inducer than lacose (1, 5 and 10 mM) for soluble form
expression.In conclusion,Theproduction ofsolublePED antigen
is highestwith 0.1 mM IPTG,induction OD600 0.6,expression
temperature15℃,harvesttime24h forrGST-COE and0.1mM
IPTG,induction OD600 0.6,expression temperature 15℃,harvest
time12hforrGST-S1D expressionrespectively.
In chapter2,we selected pTf16 chaperone system,expression
temperature 15℃,0.1 mM IPTG as inducer for expression in
fermenter level. Then we screened culture medium, growth
temperature, induction time and L-arabinose concentration.
Maximum OD600 ofsynthetic medium is two times higherthan
thatofLB broth because glucose in synthetic medium is main
carbon and energy source. Therefor, we selected synthetic medium forhighcelldensity.Todecideinductiontime,wetested
growth curve at30 and 37℃.we selected two log phase for
induction (early-log phase atinduction OD600 3∼4 and mid-log
phaseatinduction OD6007∼8.Theeffectofgrowth temperature
30 and 37℃ on theefficiency oftheinduction wasexamined at
induction OD600 3∼4 and OD600 7∼8.The production of PED
antigen was highest at growth temperature 30℃ at both
induction OD600 3∼4 and 7∼8.Then we compared induction
OD6003∼4 and 7∼8 atgrowth temperature 30℃.The induction
OD600 3∼4 was betterthan induction OD6007∼8 forexpression
ofprotein atgrowth temperature 30℃.We needed to compare
theprotein expression among 0.5,1.0and2.0mg/mlL-arabinose
at30℃ with induction OD600.3∼4.Soluble form expression of
rGST-COE wasthehighestwith0.5mg/mlL-arabinose.Finally,
the growth temperature 30℃,induction OD600 3∼4,0.5 mg/ml
rGST-COE expression in fermenter. In addition, the optimal harvesttime is 15 hr.The cells produced by using optimized
conditions were lysed using sonication and the purified by
GSTrapcolumn.Concentration ofpurifiedsolubleform protein at fermentation levelwas 53.8 mg/l.Finally,purified soluble form
proteinyieldwasenhancedby10timesfrom flasklevel(5mg/l)
to fermentation level (53.8 mg/l) (Figure 24). ESI result confirmed thatthe amino acid sequence ofthe purified protein
matchedwiththesequenceofrGST-COE.
VI
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t
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ur
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t
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VI
I
.Summar
y i
n Kor
ean
PED(돼지유행성설사병)은 전염성이 매우 강하며 국내뿐만 아니라 전 세계적으로 돼지농장에 상당한 경제적인 피해를 주는 질병이다. 본 연구의 목표는 E.coli발현 시스템을 이용하여 가장 적은 비용 으로 가장 많은 양의 PED 백신을 생산하는 것으로써 가장 경제적 인 PED 대량생산공정 시스템을 구축하는 것이다.먼저 작은 규모의 플라스크 수준에서 기본적인 조건을 잡고 그 조건을 기반으로 규모 를 확대하여 고농도의 세포를 얻기 위해 큰 규모의 발효기에서 대 량생산공정 시스템을 구축한 후 정제 조건을 확립하였다. 제 1장에서는 플라스크 수준에서 E.coli발현 시스템으로 생산된 단백질이 불용성 단백질로 생산되는 문제점을 겪게 되었는데 이것 을 극복하기 위하여 chaperone동시 발현 전략을 도입하였다.가장 먼저 다양한 chaperone 동시 발현시스템을 검정하여 pTf16chaperone 시스템이 가장 효과적인 것을 확인하였고,western blot
실험에서 anti-GST 항체를 이용하여 생산된 단백질이 목적 단백질
임을 확인 하였다.그 다음으로 chaperone유도체인 L-arabinose의
유무를 달리함으로써 chaperone 동시 발현이 목적 단백질인 rGST-COE의 수용성 발현에 효과적임을 확인하였다.단백질 발현 온도를 15,21,28,37℃로 조절한 결과 15℃에서 가장 높은 수용성 단백질 생산량을 획득할 수 있었다.발현 온도가 낮아질수록 전사, 번역과 세포 분열 속도가 느려짐으로써 수용성 형태의 생산량이 늘 어난다는 것을 검정하였다.또한 IPTG 농도를 0.1,0.4,0.7,1.0mM 에서 스크리닝하여 0.1 mM IPTG에서 가장 높은 단백질 생산량을 얻었으며,낮은 유도인자의 농도가 단백질 합성 속도를 늦추고 수용 성 형태의 생산량을 증가시킨다는 것을 확인하였다.발현 유도 시기 의 흡광도(OD600)를 0.6,09,1.2,1.5에서 스크리닝함으로써 흡광도 0.6일 때 단백질을 합성하기에 가장 건강하고 대사적으로 활발한 상 태라는 것을 확인 할 수 있었다.발현 유도로부터 12,24,36,48시
