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수용성 재조합 PEDV spi
ke단백질의 생산과
아단위 백신으로서의 항원성 검정
August
,2016
By
Nayoung Ki
m
Depar
t
mentofAgr
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cul
t
ur
alBi
ot
echnol
ogy
Gr
aduat
eSchool
Abst
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act
NayoungKim
AnimalScienceandBiotechnology DepartmentofAgriculturalBiotechnology TheGraduateSchool SeoulNationalUniversity
Porcine epidemicdiarrhea (PED)isa highly infectiousdisease occurring in 1- to2-week-old piglets.Symptomsincludewatery diarrhea and dehydration. PED is also a serious economic problem due to the high mortality rate (ZL Li.etal.,2012).In 2013, a PED outbreak in the United States caused serious economicloss.Oflate,PED hasbeenreportedinAsiancountries as well.Therefore,developmentofan effective PED vaccine is theneed ofthehour.Presented hereisa 2-partstudy,wherein wedevelopedasubunitoralvaccineconsisting oftheS1(33-591) domain of the porcine epidemic diarrhea virus (PEDV) spike protein along with an M-cell targeting ligand to effectively stimulate a mucosal immune response. We selected the E. coli(BL21)DE3,pLysS hostsystem asitiscosteffective,capable ofrapidgrowth,andiseasilymass-produced.However,thespike proteinlinkedtotheM-celltargetingligandwasexpressedasan inclusionbody.
Toovercomethischallenge,weemployedatwo-stepstrategy in Part1.The firststep involved solubilization to increase protein flexibility,whilethesecondincludedrenaturation,whichhelpedin accurate protein folding for native activity. However, solubilization and renaturation ofinclusion bodiesoften resultsin variableresults.Therefore,weoptimized theprocess to improve efficiency.Thesolubilization stepwasoptimizedby titrating urea concentration and pH,whereasrenaturation ofsolubilized protein iscomparabletoflashdilutionandpulsatiledilution.Eachmethod has its pros and cons.Finally,we performed purification ofthe protein using anion exchange chromatography, optimized by adjusting thepH.Sincethesecondary structureoftheprotein is linked to its bioactivity,the secondary structure ofthe refolded protein wascharacterized in Part2,using circulardichroism and Fourier transform infrared spectroscopy analysis. Finally, antigenicity of soluble recombinant PEDV spike protein was evaluated in vivo.Weanalyzed thelevelsofserum IgG and its subtype.Thus,solubilization and renaturation provide a method toutilizeinclusionbodiestoprepareasubunitvaccine.
Key words :solubilization,renaturation,inclusion bodies, PED, subunitvaccine,E.colisystem
Cont
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Abstract Ⅰ
Content Ⅲ
ListofTablesandFigures Ⅶ
ListofAbbreviations Ⅹ
Introduction 1
Review ofliterature 3
1.PED Virusanditsspikeprotein 3
2.Mucosalimmuneresponse 5
3.Recombinantsubunitvaccine 7
4.Recoveryofinclusionbodies 8
1)Inclusionbodies 8
2)Optimizationofsolubleproteinproductionatthehostlevel9 3)Optimizationofsolubleproteinproductionattheprotein
level 10
(1)Solubilizationofinclusionbodies 10
(2)Methodofrenaturation 12
Study 1. Optimization of solubilization and renaturation processforsolublerecombinantPEDV spikeprotein
1.Introduction 16
2.MaterialsandMethods 18
1)StrainsandPlasmids 18
2)Mediaandflaskculture 18
3)Isolationofinclusionbodies 19
4)SDS-PAGE andWesternblotassay 20
5)SolubilizationofMS1 21
6)RenaturationprocessofsolubilizedMS1 23
7)PurificationofrefoldedMS1 25
3.ResultsandDiscussion 26
1)ExpressionofrecombinantMS1protein 26
2)SolubilizationofMS1inclusionbodyprotein 31
3)RenaturationofsolubilizedMS1protein 34
4)PurificationofrefoldedMS1protein 38
Study 2.Characterization and Evaluation of solubilization and renaturation process for soluble recombinant PEDV spikeprotein
1.Introduction 44
2.Materialsandmethods 46
1)CD spectroscopy 46
(1)CD analysisofrefoldedMS1preparation 46
2)FT-IR analysis 47
(1)FT-IR analysisofrefoldedMS1preparation 47
3)InvivoassayforevaluatingantigenicityofMS1 48
(1)immunizationofmice 48
(2)MeasurementofMS1-specificantibodies 49
3.Resultsanddiscussion 50
1)CD analysistoinvestigatesecondarystructureof
refoldedMS1protein 50
2)FT-IR analysistodeterminesecondarystructureof
refoldedMS1protein 52
3)invivoimmunization 54
(1)Serum IgG titers 54
(2)IsotypeofIgG titers 56
(3)RatioofIgG2a/IgG1 59
Overallconclusion andFutureprospects 61
LiteraturedCited 65
Summary in Korea 70
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Tables
Table1.Kindofmaterialsusedinsolubilizationofinclusion
bodies 11
Table2.SolubilizationofinclusionbodiesatdifferentpH and
ureaconcentration 22
Table3.Conditionsofrenaturation 24
Table4.OptimizationofIonexchangechromatographyat
differentpHs 25
Table5.Biophysicalpropertiesandtheexpressionsystem for
MS1proteinusedinstudy 27
Table6.Totalproteinconcentrationfrom culturesgrownat
differenttemperatures 28
Table7.Comparisonoftheuseofdifferentureaconcentration
andpH forsolubilization 32
Table8.OptimalconditionsineachstepforrecoveryofMS1
Figures
Figure1.Experimentalflow chartofstudy 2
Figure2.SchematicillustrationofthePED virus 4 Figure3.Schematicrepresentationofthelymphoidelementsand antigenuptakeandrecognitioninintestinalimmunesystem 6 Figure4.Schemeofdilutionrenaturationmethod 12 Figure5.Schemeofdialysisrenaturationmethod 13 Figure6.Comparisonofoptimizationmethodforsoluble
recombinantproteininhostandproteinlevel 14
Figure7.Schemeoftheexperimentalflow chartofstudy1 17 Figure8.Schemeofthesolubilizationstepofinclusionbody 21 Figure9.Illustrationofdifferentrenaturationdilutionmethod 23 Figure10.SchematicillustrationofthetargetgeneMS1 26 Figure11.ConfirmationofrecombinantMS1proteinininclusion bodiesfrom E.coliineachgrowthtemperaturebySDS-PAGE
andWesternblotanalysis 29
Figure12.Timeoptimizationforsolubilizationinsolubilization
bufferwhichcontains2M Urea(pH12.5) 33
Figure13.ConcentrationofrefoldedMS1proteinbydifferent
renaturationmethod 36
Figure14.PurificationofrefoldedMS1proteinusingion
exchangechromatography 39
Figure15.IonexchangechromatographyusingÄKTA Protein
Figure16.SDS-PAGE analysisofelutedfractionfrom AKTA
proteinpurification. 41
Figure17.SDS-PAGE analysisofrefoldedproteinwithouturea. 41 Figure18.Schematicillustrationofremovingdenaturantfrom
solubilizedproteinforCD andFT-IR analysis 45
Figure19.CalculationmethodoftransmittancescaleinFT-IR 47 Figure20.Schematicillustrationofthecourseofimmunization
48 Figure21.FarUV CD spectraoftherefoldedMS1protein 51
Figure22.IR spectraofMS1protein 53
Figure23.MS1-specificserum IgG titers 55
Figure24.IsotypeanalysisforImmunoglobulinG (IgG) 57
Figure25.ThelevelofIgG2a/IgG1ratio 59
Figure26.Schemeofillustrationofconclusion 63 Figure27.Furtherprospectsofrenaturationstrategiesfor
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BCA :bicinchoninicAcid
CFA :completeFreund'sAdjuvant CD :circulardichroism
E.coli:escherichiacoli
ELISA :enzyme-Linked-Immuno-SorbentAssays FT-IR :fouriertransform infrared
GALT :gut-associatedlymphoidtissue IM :intramuscular
IFA :incompleteFreund'sAdjuvant
IPTG :isopropylβ-D-1-thiogalactopyranoside NALT :nasal-associatedlymphoidtissue PBS :phosphatebuffersaline
PED :porcineepidemicdiarrhea
PEDV :porcineepidemicdiarrheavirus RBD :receptorbindingdomain
S protein:spikeprotein
SDS-PAGE : sodium dodecyl sulfate polyacrylamide gel electrophoresis
TBST :tris-BufferedSalineandTween20 TGE :transmissibleGastroenteritis
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Using Escherichiacolihostsystem forexpressionofrecombinant proteinsisadvantageousdueto such high-levelexpression,fast growth,andeconomicalproductioncost.Nonetheless,heterologous proteinsarefrequently expressed in E.coliin an inactive form, asinclusion bodies.Formation ofinclusion bodiesis a challenge duetobiologicalinactivity,however,thereareseveralmerits:(i) high-levelexpression of non-native protein,(ii) easy isolation, (iii) low proteolytic degradation(Luis Felipe Vallejo and Ursula Rinas.,2004),and (iv)high levelofhomogeneity ofthe protein. We performed a two-partstudy to overcome the challenge and harnessthemeritsofinclusionbodyformation.Partoneinvolved atwo-stepstrategy ofsolubilizationandrenaturationofinclusion bodies.Solubilization isan importantstep to recovertheprotein and improve flexibility of the protein structure prior to renaturation.Nonetheless,this step requires process optimization methods, such as selection of the denaturant and its concentration,and solubilization time.Furthermore,renaturation requires efficient conditions for high yield. For instance, concentration of the denaturant and solubilized protein, the renaturation process, and selection of renaturation enhancer additives.Weoptimizedeachoftheseprocessesinthisstudy.
In Part 2, we investigated the secondary structure of the refolded targetprotein using circulardichroism (CD)and Fourier transform infrared spectroscopy (FTIR) analyses. Finally, we evaluatedtheantigenicity ofthesolublerecombinantPEDV spike protein and confirmed that the protein’s native activity was retainedviaaninvivoassayusingamousemodel.Thelevelsof IgG anditssubtypeindicatedthattheproteinhasapplicationsas anantigenfordevelopmentofsubunitvaccines.
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1.PED Virusanditsspikeprotein
Porcineepidemicdiarrhea virus(PEDV),thecausativeagentof porcine epidemic diarrhea (PED),infects epithelialcells of the small intestine in pigs. Symptoms of PED include enteritis, vomiting, watery diarrhea, and dehydration. These clinical symptoms resemble those of transmissible gastroenteritis (TGE)(Song D and Park B.,2012).However,PED is different from TGE in the target of infection. PEDV targets 1 to 2-week-old neonate piglets,resulting in a high mortality rate. However, in suckling pigs, it shows less severe clinical symptoms such as temporary weight loss or decreased milk production.
PEDV belongs to the genus Coronavirus,is approximately 30 kDa(Kocherhans R.etal,2001),and contains a single-stranded, positive-sense RNA genome. The virus contains three non-structuralproteins:1a,1b,and ORF3,in addition to four structural proteins. Spike glycoprotein (150-220 kDa) exhibits functions such as binding ofhostcellreceptorand induction of antibodies.The envelope of PEDV is composed of membrane glycoprotein(20-30kDa)andenvelopeprotein(7kDa).Thelatter
is a nucleocapsid phosphoprotein (58 kDa) that can induce cell-mediated immunity. The spike glycoprotein, which is a structuralprotein ofPEDV isimportantfordevelopmentofPED vaccine.Since it located on the viralsurface,it can regulate interactions with the host cellreceptor,mediating viralentry. Moreover,itstimulatesinduction ofneutralizing antibodiesin the host(Makadiya N.etal,2016).Thespikeprotein is divided into twodomains:S1(1-789aminoacids[aa])andS2(790-1,383aa). Themainneutralizingepitopeandthereceptor-bindingdomainlie in the S1 domain.Therefore,S1 is a promising candidate for subunitvaccinedevelopment.
2.Mucosalimmuneresponse
Themucousmembraneiscalled asthe‘mucosalbarrier’sinceit is the primary defense that protects the inside of the body against various antigens and pathogenic microorganisms that enter the body from the environment.To preventinvasion by pathogens ordiseases such asPED,vaccination isperformed at inductive sites that induce a mucosal immune response by organizedlymphoidtissuessuchasPeyer’sPatcheslocatedinthe small intestine (gut-associated lymphoid tissue [GALT]) and mucosa-associated lymphoid tissue (MALT). These sites are characterized by secretory immunoglobulin (S-IgA), which is typical of mucosal immunity(McGhee JR. et al, 1992). Additionally,these sites contain immunocompetentcells such as B lymphocytes,T helpercells,andcytotoxicT cells.Thesecells areactivated by epitopesoftheantigen afterthey arepresented by antigen-presenting cells such as macrophages and dendritic cells in GALT or MALT.Subsequently,the antigen-specific B and T cells leave inductive sites for mucosaleffector regions such as the lamina propria and the respiratory tract. Here, secretory IgA is produced after interactions between antigen-specific T and B lymphocytes. The antigen-specific S-IgA then initiates a counter attack on the foreign antigens(JerryR.McGhee.etal,1992).
Figure3.Schematicrepresentationofthelymphoidelementsand antigen uptake and recognition in intestinal immune system(MowatAM.,2003)
3.Recombinantsubunitvaccine
Vaccines are available in various forms,ofwhich,recombinant subunitvaccines utilize only an essentialepitope ofan antigen. Using a highly purified,partialtarget antigen results in high levelofsafety as compared to otherkinds ofvaccines and can elicit an immune response via immunization(Agger EM and Andersen P,2001).Additionaladvantagesduring subunitvaccine productionincludenoneedforattenuationofthevirusinanother hostsystem,which can otherwiseleadtoreducedantigenicity as well as possible contamination from the host,which in turn prevents secondary effects or adverse events caused by the contaminants(Lamphear BJ,et al.,2002).Moreover,large-scale productionandengineeringrequirementsaresimple.
In spite of these advantages,subunit vaccines have certain limitations.Due to the use of a partialantigen,the levelof immune response elicited is nothigh enough,necessitating the useofanadjuvant.Inaddition,immunityisshort-lived,requiring multiple doses(Hansson M. et al., 2000). To overcome these drawbacks,severalstrategieshavebeen explored,toenhancethe resultantimmune response,such as adjuvantincorporation and applicationofafusionpartner.
4.Recovery ofinclusion bodies
A two-stepstrategy isrequiredforrecovery ofinclusion bodies into a bio-active form.The firstinvolves solubilization ofthe inclusion bodies,while the second requires renaturation ofthe solubilizedinclusionbodies.
1)Inclusion bodies
Expression of a heterologous protein from various bacterial expression systemsoften resultsin an aggregatedform calledas inclusion bodies. Formation of inclusion bodies presents a bottleneck during production of the expressed recombinant protein,since it is biologically inactivity.However,there are many advantages ofinclusion bodies.First,high levelofpurity ofthe targetprotein in inclusion bodies (high levelofprotein expression with low level of contaminants) requires lesser amountofpurification steps.Second,isolation ofinclusion bodies from thecellsiseasy duetotheirsize(0.5– 1.3㎛)and high density.Third,formation of inclusion bodies results in a low possibility of degradation of the expressed protein by proteolysis(Singh SM and Panda AK.,2005).Severalprevious studieshavereportedtheuseofsolubilizationandrenaturationto harnessthebenefitsofinclusionbodies.
2) Optimization of soluble protein production at the host level
Several strategies have been applied to improve solubility of recombinant proteins. One example is co-expression of a chaperone,which isamoleculethatcan eitherassistin accurate folding orpreventmisfolding ofthesynthesizedprotein.Inclusion bodies are the resultofthe accumulation ofincorrectly folded protein aggregates.Therefore,using chaperone molecules is an effective method to increase solubility of the inclusion bodies. Additionalmethodsincludemodificationofcultureconditionssuch as temperature, inducer concentration, and induction time. Lowering the incubation temperature can improve solubility of recombinantproteinsasitresultsinslowingdownofintracellular processessuch astranscription and translation(Kataeva,I.etal., 2005).Asa result,lowertemperaturescan increasesolubility of inclusionbodies.Additionally,theconcentrationoftheinducercan furtherimprovesolubility ofinclusion bodies.High concentration of inducer results in a high transcription rate,increasing the intracellular burden of the recombinant protein. However,the concentration of the inducer should be accurately adjusted to reduceproduction ofinclusion bodies.Finally,theinduction time needs to be adjusted to achieve high celldensity for maximal solubilityofrecombinantprotein.
3)Optimization ofsoluble protein production atthe protein level
(1)Solubilization ofinclusion bodies
Solubilization is an essentialstep priorto accurate refolding of the protein. This step is particularly important to increase flexibility of the renatured protein obtained from the inclusion bodies(YamaguchiS.etal.,2013).Thereareseveralrequisitesto achievehighlevelofsolubility.
The first optimization factor is pH.Denaturation of inclusion bodies involves ionization ofside chains in the protein(PalmerI and Wingfield PT.,2004).Alkaline or acidic pH can lead to exposure of amino acid residues.Therefore,a combination of extremepH anddenaturantsisusedforsolubilization.
The second factor is selection of organic solutes and their concentration.Traditionally,high concentrations ofa chaotropic agent are used in solubilization,such as 6-8 M guanidinium hydrochlorideorurea.However,low concentrationsofsolubilizing agents have also been used for solubilization with extreme pH(Singh SM and Panda AK., 2005). Selecting an efficient chaotropic reagent and its concentration is specific for each proteinthatistargetedforhighyield.
Table 1.Kind of materials used in solubilization of inclusion bodies
(2)Methodofrenaturation
Optimization of renaturation to bring about accurate protein folding is a crucial step for the recovery of native protein structure. To obtain high concentration of refolded protein, renaturationiscarriedoutinvitro.
First, concentration of denaturant is reduced via dialysis, dilution,orchromatography(YamaguchiS.etal.,2013).Dilutionis thesimplestmethodforproteinrenaturationandinvolvesaddition of denatured protein quickly to the renaturation buffer,which contains low concentration to zero denaturant.In other words, concentrationofdenaturantisrapidlyreduced.Ontheotherhand, pulsatiledilutioninvolvesaddingdenaturedproteintorenaturation buffer slowly,dropwise.Thus,minimalprotein concentration in renaturationbufferisachieved(BurgessRR.2005).
Figure4.Schemeofdilution renaturation method(YamaguchiS. etal.,2013)
Different from dilution method, dialysis-based renaturation reducesconcentration ofthedenaturantgradually.However,this leads to inefficient recovery of refolded protein at high concentrations.(LuisFelipeVallejoandUrsulaRinas.,2004).
Figure5.Schemeofdialysisrenaturationmethod(YamaguchiS. etal.,2013)
Severaltypesofchromatographyhavealsobeenusedtodecrease thedenaturantconcentration.Forexample,hydrophobicinteraction chromatography, adsorption method, immobilization method, size-exclusion chromatography, and zeolite-based method(YamaguchiS.et al.,2013).These methods utilize the propertiesoftheprotein,whichresultsinefficientsuppression of protein aggregation. However, high quality renaturation using thesemethodsisaccompaniedbyhighcost.
Therefore,an idealrenaturation method is required to efficiently recovertheproteininnative,bio-activeform
Figure 6. Comparison of optimization method for soluble recombinantproteininhostandproteinlevel
(3)Purification ofrefoldedprotein
Severalmethodsareavailableforproteinpurification,suchasion exchange chromatography,based on affinity between ionizable molecule and an ion exchanger.This is an efficientmethod to improve purification sincethenativeconformation ofthe protein is preserved when it is eluted from the column.Additionally, binding capacity ofthe ion exchanger is high as compared to resins used in other chromatography methods. Moreover, the separation principle and operation of the purification cycle are simple. Though this method has many benefits, there are limitations to its use. Choosing an appropriate buffer pH is significantly importantaspH during thechromatography run can causedeformationoftheproteinstructure.Therefore,modification ofefficientloading bufferpH thatis well-suited to the protein, with high binding capacity is necessary to purify the protein.
St
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1.Introduction
Therearemanyadvantagesinusing Escherichiacoliasthehost system for production ofrecombinantproteins,butaggregation forms called inclusion bodies are frequently obtained.Inclusion body proteins are biologically inactive,however,there are some advantages with their use.For example,inclusion bodies can preventproteolyticdegradation,whileaffording high homogeneity, easeofisolation,and high-levelofexpression.In thisstudy,we used inclusion body proteinsthatweresubjectedtosolubilization and renaturation processes to enhance recovery of soluble, bioactive protein. Depending on the target protein, various strategies can be applied for solubilization and renaturation.In this study,we explored various concentrations of urea,pH of solubilization buffer, and different renaturation methods to optimizetheyield.
2.MaterialsandMethods
1)StrainsandPlasmids
E.coliBL21(DE3) pLysS(Novagen) was used for production of target gene which is MS1. Final expression plasmid was pET21(Novagen) vector which has Nde1 and Xho1 restriction sites,targetgenewasintegrated between them.Alsoitcontains ampicillinresistancegene.
2)Mediaandflask culture
LB/ampicillin broth medium :tryptone 10 g/L,yeastextract5 g/L,sodium chloride 10 g/L(Becton,Dickinson and Company, USA), ampicillin 100 mg/L(Bio basic canada inc, Canada.). Inducer:IPTG 0.3mM(Calbiochem,USA).
LB/ampicillin(Luria-Bertani)mediawasusedforbacteriagrowing and production of recombinant MS1 protein. The cells were grown at 37℃,250rpm in shaker incubator for overnight and then in same media,protein expression was induced at 37℃, 250rpm inshakerincubator4hafterIPTG induction.Also,16-20h incubation at23℃,250rpm in after IPTG induction before cell harvesting.IPTG inductionwasperformedinOD600≈0.5-0.6.
3)Isolation ofinclusion bodies
Lysis buffer1 : 50 mM Tris-Hcl(pH 8.0), 140mM Nacl. Lysozyme0.5mg/ml(Biobasiccanadain,Canada.),Lysisbuffer2 :lysis buffer1 + triton X-100 0.5%(Sigma).DNase1 500 ㎍/ml (Biosesang, Korea). MgSO₄10 mM. 10X Phosphate Buffered Saline(PBS)werepurchasedfrom Hyclone(Utah,USA)
E.coli cells were harvested at 4℃, 6000rpm for 5min by centrifuge.Andthenwetpelletswereresuspendedin 1X PBS 50 ml 3 times for washing.Then,they were kept in –70℃ deepfreezer after removing supernatant.In the next day,cell pelletwas thawed for10min and then re-freeze itfor20min in –70℃ deepfreezer. For easy cell disruption, this cycle was repeated 3 times.Afterthisstep,cellpelletwasresuspended in lysis buffer1 with lysozyme.And then incubation was carried outat37℃,250rpm inshakerincubatorfor1-2h.AddingDNase1 andMgSO₄during thisincubationtime.Afterthat,sampleswere centrifuged at 4℃, 14000rpm for 10min to discard the supernatant.Then,cellpelletswereresuspendedinlysisbuffer2 with 0.5% triton x-100and itwasincubated at37℃,250rpm in shaker incubator for 1h. Finally, cells were disrupted by sonication and then thesesampleswerecentrifuged at12000rpm for10min.Forrecovery ofpurified inclusion body proteins,lysis
bodies were used a series of steps for recovery of soluble protein.So,freeze drying of inclusion bodies was involved in purificationstepforincreasingperiodofuse.
4)SDS-PAGE andWestern blotassay
SDS-PAGE wasconducted using 5% stacking gelsand 15% or 12% resolving gels underreducing condition.Allgels were run 80V for40min in staking gels and 120V for80min in resolving gels.Proteinswereloadedon gelsstainedby Coomassieblue.In case of western blot assay,it was performed using anti-His antibodiesto confirm existenceofrecombinantproteins.On 15% resolving gels,proteins were separated at120V for 80min and transferred onto a nitrocellulose membrane(GE healthcare) by Xcell blot module transfer(Invitrogen) at 13V for 90min. 5% skim-milk in TBST wasused forblocking atroom temperature for1h.Anti-His antibody was treated atthe rate of1:1000 in 4℃ forovernight.Anti-mouse IgG conjugated with horseradish peroxidase anti-bodies were used as secondary-antibodies.They were treated atroom temperature for1h fordetection oftarget protein.
5)Solubilization ofMS1
PurifiedMS1inclusion body proteinsweresolubilizedin various concentration of urea(sigma) and different pHs in 20 mM Tris-HClsolubilization bufferatroom temperature.Concentration ofurea in solubilization buffers varies from 0 M to 8 M and they had two ofeach type in pH.pH 8.5 and pH 12.5.MS1 inclusion bodies 5mg were solubilized at 1ml of each solubilization buffer.Concentration of solubilized proteins were analyzed by a BCA assay.Finally,yield ofsolubilization was expressedinpercent.
Table 2.Solubilization ofinclusion bodies atdifferentpH and ureaconcentration
6)Renaturation ofsolubilizedMS1
Renaturation buffer :Urea (sigma,finalurea concentration of buffer:0M or2M),5% sucrose(sigma),20mM Tris-HCl(pH 8.5)
Solubilized MS1 protein was refolded by two types of renaturation methods.The first method was a flash dilution. Solubilized MS1 was diluted rapidly in renaturation buffer and then it was incubated overnight at room temperature with stirring 100-200rpm.Thesecond method wasapulsatiledilution. Denatured protein was added to renaturation bufferslowly,drop by drop.So,timeintervalswereexistbetween dropped samples. Also this method was performed at room temperature with stirring 100-200rpm. pH of all renaturation buffer was 8.5. Concentration of refolded proteins were analyzed by a BCA assay.
7)Purification ofrefoldedMS1
Equilibrium buffer :Urea 2 M(sigma),5% sucrose(sigma),20 mM Tris-HClinvariouspH
Elution buffer : 1 M Nacl(Biosesang, Korea) was contained equilibrium buffer,Hitrap Q FF column 1 ml(GE healthcare biosciencesUppsala,Sweden).AKTA proteinpurificationsystem.
Refolded protein samples were centrifuged at 17000rpm for 20min.And then cleared supernatant was used as sample of anion exchange chromatography.First,column was equilibrated with equilibrium bufferand bounded sample protein was eluted by elution buffer.Renaturated MS1 protein was observed by stages of increasing pH(pH 7.0- pH 9.0). Concentration of purifiedproteinswereanalyzedbyamicroBCA assay.
Table 4. Optimization of Ion exchange chromatography at differentpHs
3.ResultsandDiscussion
1)Expression ofrecombinantMS1protein
Thetargetgene,designatedasMS1comprisedacombinationof M cell-targeting peptideattheN-terminaland 33-591aaofthe S1 domain ofthe PEDV spike protein,as shown in Figure 9. The strain of PEDV used was PPⅣ,cloned in recombinant plasmidpET21vector.Additionally,6His-tag fusion protein was fusedwiththeconstruct.
Table 5.Biophysicalproperties and the expression system for MS1proteinusedinstudy
Though neutralizing epitopes can induce a specific immune response, the resultant antibody response is not as strong compared with that achieved using native S1 domain.Several previous reports have utilized adjuvant to increase immunogenicity. Nonetheless, use of whole S1 domain can achievehigherimmunogenicity ascomparedtoan artificial,short epitope owing to its resemblance to the native structure. Moreover,SP1 region includes a receptor-binding domain (RBD) and neutralizing epitope,which suggests that the antigenicity afforded willbe higherthan thatachieved by vaccination with theepitopealone.Moreover,thisregion may bemoresimilarto the native structure of S1 domain as compared to the short epitope obtained afterthe renaturation process,since itcontains almostthefulllength oftheS1 domain.Thus,thesimilarity in structuremayleadtonative-likeimmunogenicity.
Severalrecentstudieshavebeen reported regarding RBD-based subunitvaccines.LiF etal.and He Y etal.investigated the effectivenessofan RBD-based vaccine againstSARS virusand reported that neutralizing antibodies against SARS virus are induced.Infact,RBD-basedvaccinestrategieshavebeenusedin thecontextofSARS virusbutnotin caseofPED.Theresults of this study suggest that RBD of PEDV is presented to candidateimmunogensbytheRBD-basedvaccine.
Table 6. Totalprotein concentration from cultures grown at differenttemperatures
Figure11.Confirmation ofrecombinantMS1protein in inclusion bodies from E.coliin each growth temperature by SDS-PAGE and Western blotanalysis.(A)Growth temperature :23℃.(B) Growth temperature :37℃.*N.I:non-induction ofIPTG,S : soluble fraction,IB :inclusion body.M :marker,IB recovery : inclusionbodiesweretreatedbyfreezedrying.
Figure 10 shows the protein expressed in E.coliBL21(DE3). pLysS and0.3mM IPTG wasaddedtoinduceproteinexpression at23°C for16-20h and 37°C for4h.Asa result,recombinant MS1 protein was expressed in the inclusion body form ateach growth temperature.Additionally,concentration of totalprotein was higher at 23°C than at 37°C. However, lower growth temperature cannot prevent inclusion body expression. MS1 inclusion bodies obtained atgrowth temperature of23°C yielded 0.03 g/mlaggregation proteins,which was higher totalprotein concentrationthanthatobtainedat37°C (Table6).
Using inclusion bodiesastheantigen forpreparation ofsubunit vaccine has several benefits. First, the E. coli system can produce large quantities ofaggregated protein and the isolation method is easy and cost-effective.Besides,there is no need to employ time-consuming techniquesofantigenpurification.Dueto itshigh homogeneity,the antigen can beeasily prepared.Ifthe immune-dominantepitope is notsequestered in the aggregated form,it holds promise as a candidate antigen for use as a vaccine.Kesik M etal.and H Yang etal.usedinclusion bodies as vaccine antigen and demonstrated antigenicity via antigen-specific antibody titer.However,P1 and P2 inclusion bodies do notinduce immune response,as shown in previous studies. Therefore, in this study, we performed a series of solubilization and renaturation stepsto solubilizeand recoverthe
bioactiveform.Furtherstudiesarenecessary todevelopmethods touseinclusionbodyproteinsforimmunization.
2)Solubilization ofMS1inclusion body protein
Solubilization yield was analyzed by bicinchoninic acid (BCA) assay using supernatantofsolubilization samples and presented as a percentage. The highest yield, obtained with 20 mM Tris-buffer and 2 M urea at pH 12.5, was in excess of 90%(Table 7).However,there was no significantdifference in yield of solubilized inclusion bodies obtained using different growth temperature.Moreover,higherconcentration ofurea did notresultinincreasedsolubilization in20mM TrisbufferatpH 12.5. Although further addition of urea showed increased solubilization in 20 mM Tris-bufferatpH 8.5,these conditions werenotaseffectiveasapplyingpH 12.5.Using2M ureaatpH 12.5, the average yield of denatured proteins obtained from inclusion bodies prepared at 23°C and 37°C,was 4.7 and 4.5 mg/ml,respectively.Thus,acombination ofalkalinepH andlow concentration ofurea facilitated dissolution ofthe targetprotein, MS1 forincreased solubilization.Ofnote,the high pH was an essentialrequisiteforeffectivesolubilization,however,theusage ofureawasalsoanimportantfactortoincreasesolubility
Table 7.Comparison ofthe use ofdifferenturea concentration and pH for solubilization.(A),(B) MS1 inclusion body proteins were solubilized in various solubilization buffers which contain differentureaconcentrationandtwotypesofpH.Thesolubilized protein concentration in thesupernatantwasanalyzed by aBCA assay.Percentsolubilityiscalculatedbytheresultofit.
Time optimization ofsolubilization was performed ateach time point0.5h,1h,3h,5h,12h,and 24h using 20 mM Tris buffer with 2M ureaatpH 12.5.Theresultshowed thatsolubilization was done at 3h. Additional time can not lead to better solubilizationunderthiscondition.
Figure 12.Time optimization for solubilization in solubilization buffer which contains 2 M Urea(pH12.5). Each sample was solubilized in solubilization buffer which contains 2M urea concentration and pH12.5.At0.5,1,3,5,12,24 time points,a solubilized protein concentration in thesupernatantwasanalyzed byaBCA assay.(n=3,Errorbarrepresentsstandarddeviation)
3)Renaturation ofsolubilizedMS1protein
Estimation ofrefolded MS1 protein concentration showed that flash dilution renaturation process achieved higher yield as compared to pulsatile dilution renaturation. Additionally, the efficiency in recovery of inclusion bodies was proportionalto solubility.Thus,maximum concentration ofrefoldedproteinswas obtainedfrom solubilizationin 20mM Triswith2M ureaatpH 12.5.Previous results from solubilization ofinclusion bodies and recoveryefficiency indicatethattherewerenonotabledifferences between growth temperatures used.Samples were denatured in solubilizationbuffercontaining 20mM Triswith2M ureaatpH 12.5 and then refolded at a high rate through flash dilution renaturation, to obtain correctly refolded MS1 target protein. Maximum concentration ofrefolded protein using flash dilution renaturation strategy was0.21 mg/ml.Additionally,glycerolwas not effective as a renaturation stabilizer (data not shown), whereas 5% sucrose helped increase stability of renatured proteins.As shown in Figure 13,presence of urea improved renaturationefficiency.
Figure13.ConcentrationofrefoldedMS1proteinbydifferent renaturationmethod.(A)Targetgrowthtemperature:23℃.(B) Targetgrowthtemperature:37℃ (A,B)Eachsampleswere solubilizedin0M,2M urea(pH12.5),8M urea(pH8.5)andthen theyweredilutedineachrenaturationbufferthroughflash, pulsatiledilutionmethod.Concentrationofrefoldedproteininthe supernatantwasanalyzedbyaBCA assay.(C)Comparisonto concentrationofrefoldedproteinwhichexpressedindifferent growthtemperature.(n=3,errorbarrepresentsstandard deviation;*p< 0.05,**p< 0.01,***p< 0.001,ANOVA of repeatedmeasuresone-wayANOVA.)
Urea was used as a denaturant to obtain high level of solubilization athigh concentrationssuch as8.0M,whereaslow concentrations such as 2 M served as a stabilizer for the refolding process.Thepresenceofadenaturantsuch asureaor GdmHClcan improve renaturation efficacy(Hevehan DL and De Bernardez Clark E,1997.YamaguchiS.etal.,2013).Although information of this mechanism is limited,the commonly used explanation is thatchaotropic agents preventthe interaction of protein surface, thus inhibiting protein aggregation. This explanation was confirmed by renaturation yield.Efficiency of renaturationatureabufferconcentrationof2M wassignificantly higher than that in absence of urea.However,this strategy cannot be applied to allproteins.The presence of chaotropic agents induce aggregated forms in competitive reaction between refolding intermediates and aggregation(Yamaguchi S. et al., 2013).Moreover,sucrose,glycerol,and sugars are employed as stabilizersin therenaturation processforhigh yield ofrenatured proteins.However,its mechanism is controversialand usage of differentkindsofstabilizersalsoenhancesformation ofinclusion bodiesin thein vitro refolding process.Forsuccessfulrecovery of solubilized proteins, time consuming approaches for optimization ofproper refolding conditions are to each target protein
4)Purification ofrefoldedMS1protein
Anionexchangechromatographywasusedinthisstudy,to purifyMS1,wheretherunningbufferhadapH higherthanthe isoelectricpointofMS1,elsetheproteinwouldnotadheretothe resin.Theisoelectricpointofthesamplewas5.19,thereforea runningbufferpH rangeof7.0– 9.0wasusedtoidentify optimalconditionstoobtainhigheryieldofpurifiedprotein.We observednotabledifferencesinquantityofpurifiedproteinacross thepH range,withhighestpurificationatpH 8.5andaveragenet recoveryofpurified,correctly-refoldedproteinof1.75mg/ml.
Figure14.PurificationofrefoldedMS1proteinusingion exchangechromatography.(A)Concentrationofpurifiedtotal protein waseluted1M NaClelutionbuffer.Eachelutionfraction volumeis1.25ml.(B)Thenumberofelutionfractioniseight sampleandonlyfiveelutionsampleisloadedSDS-PAGE.Total purifiedMS1proteinwasanalyzedbyamicroBCA assay
throughelutionfraction.(n=3,errorbarrepresentsstandard deviation;*p< 0.05,**p< 0.01,***p< 0.001,ANOVA of repeatedmeasuresone-wayANOVA data.)
Ion exchange chromatography was performed using ÄKTA Protein Purification Systems with 1 mlHitrap Q FF column. Starting material, sample flow-through, washing fraction, and elution fractions were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels(Figure 16). Moreover, another bottleneck encountered during solubilization and renaturation procedures using chemical solvents was re-aggregation while removing denaturants such as urea. However,in this case,sufficient amounts of refolded protein remainedinthesolubleform showninfigure17.
Figure15.IonexchangechromatographyusingÄKTA Protein PurificationSystems.
(A)Startbuffer:20mM Tris,2M Urea,5% Sucrose(pH8.5), Elutionbuffer:startbuffer+1M NaCl.Gradientvolume:20 ml,Flow rates:1ml/minusingHitrap1mlcolumn.
Figure16.SDS-PAGE analysisofelutedfractionfrom AKTA proteinpurification.(A)SDS-PAGE analysisofsolubilized
fractionfrom 2M ureaatpH12.5inTrisbuffer.Lane1:Protein MW marker.2:solubilizedfraction.(B)SDS-PAGE analysisof elutedfractionfrom AKTA proteinpurification.Lane1:Protein MW marker.2:refoldedprotein,3:Sampleflow throughfraction, 4:washingfraction,5-9:elutedfraction.
Figure17.SDS-PAGE analysisofrefoldedproteinwithouturea. Allelutedfractionsarepooledbyultra-centrifugalfilters.
4.Summary
E.colias host cellexpression system is an idealchoice for producing target protein owing to its cost-effectiveness,rapid growth,and high-levelofexpression oftargetprotein.Despite these advantages,a crucialobstacle to successfulexpression of protein in the E.coli host system is the formation of inert aggregates as called inclusion bodies.In this study,our target proteinMS1wasexpressedininclusionbody form.Toovercome this limitation, solubilization and renaturation process were performed for recovery of the protein’s biologicalactivity.To identify idealconditions for solubilization and renaturation,we investigated solubilization ofMS1 inclusion body proteins using different solubilization buffers,different concentrations of urea,
andvaryingpH.
Finally,there were two requisites for maximalsolubility.The firstone was urea and the second,alkaline pH.Combination of these conditions resulted in high solubilization of target MS1 protein.For renaturation,flash dilution achieved high yield of correctly-refolded protein compared with pulsatile dilution.For purification,renatured proteins were stabilized at pH 8.5.The BCA ormicroBCA assaywasusedtodetermineproteinyield.
Table 8.Optimalconditions in each step forrecovery ofMS1 inclusionbodies.
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1.Introduction
Three-dimensionalstructureofproteinisnecessarytounderstand itsbiologicalfunction.Forthisreason,itisimportanttoestimate structuralconformation of refolded protein to determine if its nativebiologicalactivity hasbeen recovered.Severalmethodsto determine protein structure are available,such as CD analysis, nuclearmagnetic resonance spectroscopy,X-ray crystallography, and FTIR spectroscopy.One of the best ways to investigate structuralelements is CD spectroscopy,which shows structure and folding characteristics ofprotein(Norma J Greenfield,2006). FTIR spectroscopy isanotherexcellentmethodtoanalyzeprotein structure(Kong Jand Yu S.2007).In thisstudy,weused both, CD and FTIR to evaluate the secondary structure ofrefolded protein.We confirmed thatMS1 protein was correctly refolded and it behaved similar to its native soluble form via in-vivo immunization of mice.By confirming antigenicity in terms of titersofserum IgG and IgG subtype,weconcludethatrefolded MS1proteinisacandidatesubunitvaccineagainstPED.
Figure18.Schematicillustrationofremovingdenaturantfrom solubilizedproteinforCD andFT-IR analysis.
2.Materialsandmethods
1)CD spectroscopy
Preparationofsample:Removalofureafrom refoldedproteins Total 2 M of urea concentration in sample solution was removed by Amicon ultracentrifugalfilters(Milliporeireland Ltd, Ireland).Purified refolded protein samples were centrifuged for 15min atroom temperature using centrifugalfilters.And then, theyweredilutedwith1X PBS byaratioof1/10at4times.
(1)CD analysisofrefoldedMS1preparation
:CD measurementswereperformed atroom temperatureusing Chirascan plus spectrophotometer (AppliedPhotophysics, UK). Concentration ofprotein was0.8mg/ml(which proteinscamefrom 23℃ofgrowth temperature)and 0.295mg/ml(which proteinscame from 37℃of growth temperature) in 1X PBS buffer. Its concentration was analyzed by a BCA assay.Aqueos solution samples were recorded in 190-260nm path length.The mean of Deltaellipticitieswascm²·dmg/ml⁻¹calculated721aminoacid.
2)FT-IR analysis
Preparation ofsample :Removalofurea from refolded proteins andfreeze-drying
Procedure ofremoving denaturantwas equalto preparation of sample used forCD analysis.And then,aftereliminating urea, samples were underwent freeze-drying.Purified samples came from eachgrowthtemperatureat23℃and37℃.
(1)FT-IR analysisofrefoldedMS1preparation
: Prepared samples were estimated on ATR Acc.(window ZnSe/diamond) - Nicolet 6700(Thermo Scientific, USA) spectrometer was used for collecting spectra. During measurement,refolded proteins were analyzed as freeze dried pelletform in room temperature.Measurementresolution value
was8cm-1and itsnumberofscanswas32.TheWavenumber
3)Invivoassay forevaluating antigenicity ofMS1
(1)immunization ofmice
7-week-old BALB/C female mice(18 mice)were divided into 3 groups becoming 6 mice per group following the policy of laboratory animal facility (Laboratory Animal Center, Seoul NationalUniversity,Korea).Totalexperimentalduration was 6 weeks.The purified refolded MS1 antigens (50 ㎍/mouse)were subcutaneously injected 2 times at 0 and 14 days(2-weeks intervals). Priming injection was given by purified MS1 emulsified in Complete Freund’s adjuvant(50 ㎕/mouse) and boosterimmunization was given by purified MS1 emulsified in Incomplete Freund’s adjuvant(50 ㎕/mouse).Blood sampling was conducted 0,14,28 days.Mice weresacrificed at28 days after blood sampleswerecollected.Collected blood sampleswereused analysisoftotalIgG anditssubtypes.
(2)MeasurementofMS1-specificantibodies
ThelevelsofMS1-specificIgG,IgG1andIgG2aantibodies in serum weredeterminedby ELISA.96-wellimmunoplates(SPL, Korea) were coated at 1 ㎍/ml of purified refolded MS1 recombinantprotein in 50mM carbonate–bicarbonatebuffer(pH 9.6)at37℃ for2h incubation then washed with 1X PBS at3 times.And then immunoplates were blocked with 1%(weight volume)BSA atroom temperaturefor1h then washed with 1X PBS at 3 times.1:2500 diluted serum samples were add into wellsthenincubationat37℃ for2h.Afterincubation,eachwells were washed with 200 ㎕ of PBST(PBS containing 0.05% of Tween20)at3times.
HRP (HorseRadish Peroxidase)-conjugatedgoatanti-mouseIgG, IgG1,orIgG2a (Santa Cruz Biotechnology,USA)was added to each wells atroom temperature for 1h.After then,designated wells were washed 200 ㎕ ofPBST at3 times.TMB solution (Sigma–Aldrich,USA) was add to the wells for substrate of HRP.Afterincubation atRT for5min,thereaction wasstopped
by 100 ㎕ of 0.16 M H₂SO₄.The absorbance value was
determined by GRL 1000 Microplate Reader (General Labs Diagnostics,USA).TheresultsofELISA wereexpressed asOD valuesmeasuredat450nm.
3.Resultsanddiscussion
1) CD analysis to investigate secondary structure of refoldedMS1protein.
To examine the secondary structure ofrefolded MS1 protein, weperformed CD analysis.At190-260 nm ofCD spectra,there were no significant differences in secondary structure at both growth temperatures. The proportion of α-helix was 23.2% (inclusion body proteins isolated from E.coligrown at23°C), 23.6% (inclusion body proteins isolated from E.coligrown at 37°C). Additionally, proportion of β-antiparallel structure was 25.7% (23°C) and 25.0% (37°C) of refolded MS1 proteins. Additionally,the β-parallelcomponent ratio was 10.4% (23°C) and 10.2% (37°C),while the remaining portion consisted of a random coil structure. The CD spectra results suggest that varioussecondarystructuresweremixedwithintherefoldedMS1 protein, where β-sheets (β-antiparallel and β-parallel) represented a higher proportion than any other secondary structure(figure21).
Moreover, the CD spectrum of the target protein was very similartotheS35fragment,located attheN-terminaloftheS1 domain ofTGE virus(Transmissible Gastroenteritis Coronavirus) spike protein(JReguera ,etal.,2011).This region binds to the
enteric receptormolecule.Thus,ourresults provide evidence of effective antigenicity when refolded MS1 is used as a subunit vaccineforPED
Figure 21.Far UV CD spectra of the refolded MS1 protein. Afterpurification,urea was removed by Ultra centrifugalfilters at4 times.Protein concentration is 0.8mg/ml(23℃),0.295(37℃) andanalysiswasconductedwithpathlength190-260nm.
2) FT-IR analysis to determine secondary structure of refoldedMS1protein.
Theinfrared responsewasestimated by FTIR ofrefolded MS1 protein isolated from E. coli grown at 23°C and 37°C for comparison ofsecondary structure obtained atdifferentgrowth temperatures. The second derivative spectrum did not show significant difference between the two conditions.In addition, solubleformscoexisted with insolubleprotein aggregatesin both cases.Thepresenceofinclusionbodyproteinsweredemonstrated by theIR spectrum peaksat1065.47and 1058.42 cm⁻¹,whereas the formation ofsecondary structures were monitored in the IR spectrum.Averagepeaksat1637.85cm⁻¹(23°C)and1637.94cm⁻¹ (23°C)indicated the presence ofβ-sheets structures within the targetsolubleproteins.
In comparison with previous CD analysis data, β-sheets structureswere observed in both cases.However,therewasno absorption at 1648-1657 cm⁻¹, which is indicative ofα-helix structure. Put together, we concluded that the native-like secondary structure was presentin the refolded targetprotein, which implies thatsolubilization and renaturation processes had improvedrecoveryofMS1aggregates.
Figure22.IR spectraofMS1protein.Proteinsamplecamefrom Inclusionbodieswhichgrownat23℃and37℃.Thenpurified samplewasfreeze-driedwithoutsolubilizedsolvent.
3)invivoimmunization
(1)Serum IgG titers
Invivoimmunizationfor4weekswasperformedusing 18mice, to confirm the antigenicity ofrefolded MS1 targetprotein.We hypothesizedthatimmuneresponseswereinducedbytherefolded protein becauseitsbio-activity wasrestored by therenaturation process.To validate ourhypothesis,we performed immunization with subcutaneousadministration ofrefolded MS1with Freund's complete adjuvant (CFA),phosphate-buffered saline (PBS) and commercialPED vaccinewith CFA adjuvant.Afterimmunization, the renatured MS1 specific immune response was evaluated by enzyme-linked immunosorbent assay using mice serum. As shown in Figure 23,the mice immunized with refolded MS1 protein with CFA adjuvant showed significantly high levelof serum IgG titer compared with PBS group and PED vaccine group at4weeks.Titerofpositivecontrolwaslowerthan that of MS1 group because the antigen used in commercialPED vaccine was derived from DR13 strain;however,MS1 used in thisstudy wasderivedfrom PPIV strain.Duetostrainvariation, IgG from thepositivecontrolgroup did notreactwith theMS1 protein.Thus,aggregated MS1 protein had successfully refolded and the denaturation-renaturation procedure used in this study effectivelyrecoveredbio-activityofaggregatedprotein.
Figure23.MS1-specificserum IgG titersat2and4week.Each dotdenotesanti-MS1antibodytitervaluefrom individualmouse andthebarrepresentsaveragemean(n=6ineachgrouptested). Allvaluesrepresentthemeans± SD.(*p< 0.05,**p< 0.01,
(2)IsotypeofIgG titers
To further investigate in vivo effects of refolded MS1, we analyzed the levels of MS1-specific serum isotype of IgG. Immune responses from T-helper cell1 (Th1) induces IgG2a, whereas IgG1 is obtained from T-helper cell2 (Th2)immune activity(Mosmann TR and Coffman RL.,1989).Therefore,the levels of IgG2a and IgG1 are indicative of both immune responses.Subsequently,we confirmed immune responsiveness induced by refolded MS1 protein in terms of these antibody titers.As shown in Figure 24,the levelofIgG2a was higher with PBS and commercialPEDV groupat4weeks.Additionally, the titerofIgG1 was higherthan thatofothergroups butthe results did not show significant difference at 4 weeks.At 2 weeks, there was little difference among the groups. In conclusion,refoldedMS1caninducebothTh1immunitytypeand Th2immunity type.Moreover,IgG2aandIgG1wereproducedin balanced way,indicating thatbalanced immuneresponsesdriven from Th1andTh2wereinducedby refoldedMS1targetprotein, whichunderwentsolubilizationandrenaturationprocess.
Figure 24.Isotype analysis for Immunoglobulin G (IgG).(A) MS1- specific serum IgG1 at2 and 4 week.(B)MS1-specific serum IgG2aat2and4week.Thebarrepresentsaveragemean (n=6 in each group tested).Allvalues representthe means ± SD.(*p< 0.05,**p< 0.01,***p< 0.001,one-wayANOVA)
Spike protein ofPED virus is a crucialtargetfor developing effective subunit vaccine,since it plays a significant role in interaction with aminopeptidase N to allow viralentry into the host and induces neutralizing antibodies after invasion to host(Chang SH et al., 2002).However,the S protein shows variousgeneticvariation sousageofcurrentPED vaccineshave limitations of effectiveness.In Korea,two types of attenuated PEDV vaccine are used:SM98-1 strain is used to prepare IM vaccineandDR-13isusedtoprepareoralvaccine.Bothvaccines have shown low protection efficacy during epidemic PED outbreaks(Lee C.2015).An explanation for this is nucleotide variationsbetween PEDV strainsin Korea.Phylogeneticanalysis ofS protein shows a large distance between the vaccine and field strains.DR-13 strain used in this study is included in classicalstrains ofGenogroup 1 butPPIV,which is ourtarget strain isolated from the field is classified into Genogroup 2(Lee C.2015).Duetotheirgeneticheterogeneity,titersofIgG andits subtypesfrom commercialPEDV DR-13 vaccinedo notrespond to MS1 target derived from the PPIV strain. These results suggestthatdeveloping effective next-generation PEDV vaccine requiresisolationoffieldvirusstrains.
(3)RatioofIgG2a/IgG1
The ratio of IgG2a / IgG1 is used as an indicator to judge protective immune response.Ifthe ratio is higherthan 1,Th1 responseissuperiorto Th2response.Whereas,iftheratiois< 1 itimplies a Th2 response.As shown in figure 25,the ratio
valueofMS1was1,implying thatboth,Th1andTh2immunity
responseswereinduced in abalanced way,becauseofCFA and
IFA.
Figure25.ThelevelofIgG2a/IgG1ratioat4week.Each
bardenotesIgG2a:IgG1titercalculatedbyOD valueofIgG2a andIgG1(n=6ineachgrouptested).Allvaluesrepresentthe means± SD.
4.Summary
Characterization of refolded protein using CD and FT-IR was essential for assessment of correct renaturation. Although secondary structure is not direct evidence,the existence of a native-like secondary structure is pivotalevidence ofsuccessful protein recovery (AmiD.etal.,2006).Wehypothesized thatour solubilization and renaturation process can effectively recover targetinclusion body protein MS1 and through CD and FT-IR analysis,wedemonstrated thattheprotein obtainedexhibited the requisitesecondarystructure.
Moreover,the finalaim ofthis study was in vivo antigenicity. The in vivo immunization assay is very meaningful because serum IgG titersaredirectevidenceofcorrectrenaturation.The results from the in vivo experimentshowed thatrefolded MS1 targetprotein induced immune response,implying thatrenatured MS1isan effectiveinducerofimmuneresponsesand itcan be usedasasubunitvaccinetopreventPED.
Over
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The s1 domain ofthe PEDV spike protein is a majortarget protein forvaccination becauseitislocated on theviralsurface. This allows itto regulate interactions with hostcellreceptors then inducing neutralization antibodies in the host.Using this region toprepareasubunitvaccinetopreventPED,weselected hostcellsystem E.coliforproduction oftargetprotein because itsadvantagessuch asrapid cellgrowth,costeffectiveness,and high expression level of target protein. However, our target recombinantprotein MS1 was expressed in aggregate form.To overcomethisbottleneck and takeadvantageofinclusion bodies, we performed denaturation and renaturation progress recoverits bioavailability.During developmentofrenaturation methods,we wanted to achieve high concentration ofrefolded protein atthe same time.In this study,we developed a seriesofprotocolsto obtain our targetinclusion body protein MS1 in soluble form. First,weusedvariousureaconcentrationsandtwopH conditions tosolubilizeprotein aggregatesand identify optimalsolubilization conditions.Combination of2 M urea and alkaline pH (pH 12.5) wasfoundidealforthispurpose.However,highpH leadstohigh yield ofsolubilization,mild concentration ofurea also improves concentration of denatured protein. Secondly, flash dilution renaturation system was effective as compared to pulsatile
dilution system.Overall,different inclusion bodies obtained at differentgrowth temperaturesdidnotshow significantdifference. Lastly,ionexchangechromatography wasusedtopurify refolded protein,atpH 8.5toobtainhighyieldoftargetprotein.
In Study 2,we characterized and evaluated refolded MS1 to confirm recovery ofits bioavailability.To confirm this strategic point,we used CD and FT-IR analysis to determine protein secondary structure, which revealed native-like secondary structures. CD analysis indicated the presence of α-helical structure, β-sheet structure, and random coils. Unlike CD analysis,inFT-IR analysissuggestedthatonlyβ-sheetstructure waspresentinthesolublefractionandaggregatesform wasalso existed in targetsoluble sample.However,similar to Study 1, therewereno notabledifferencesin inclusion bodiesprepared at differentgrowth temperatures.The in vivo immunization assay evaluated antigenicity of soluble recombinant spike protein of PEDV in mice for 4 weeks,using subcutaneous injection of targetantigen twice atan intervalof2 weeks.As shown in terms ofserum IgG titers,immune responses were induced by refoldedMS1specifically.Moreover,thetitersofIgG2aandIgG1 as isotype ofIgG,which representTh1 type immune response and Th2 type immune response,respectively,showed thatMS1 canleadtobalancedimmuneresponse.AlthoughthetitersofIgG anditssubtypeconfirmedantigenicity,thereisaneedforfurther
study to compare with the aggregated form ofMS1.Through comparativein vivo study between inclusion bodiesand refolded form, existence of bio-activity by form of protein can be confirmed.
In conclusion,the renaturation pathway developed in this study effectively recovered the native bioactivity and allowed accurate refolding of solubilized MS1, which can be considered as a potentialcandidateasasubunitvaccinetopreventPED.
Although the expression ofinclusion bodies is stilla challenge, design of suitable solubilization and renaturation protocols can helpovercomethislimitation.
Figure 27. Further prospects of renaturation strategies for reproductionofsolubleproteinfrom inclusionbodies
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돼지유행성설사병(PED)은 PED 바이러스의 감염에 의한 질병으로서 수양성 설사 및 구토를 유발하여 신생자돈에서의 높은 치사율을 보 이며 동시에 이로 인한 경제적 피해를 입히는 전염병이다.본 연구 에서는 PED 질병을 예방하고자하는 서브유닛 백신개발의 일환으로 E.coli발현 시스템에서 불용성 단백질로 생산되어지는 PEDV spike protein 33-591a.a 까지의 영역을 단백질 수준에서 가용성 형태로 얻고자 하였고,이의 구조적 특징을 규명하고 생체 내 면역 실험을 통하여 항원력을 평가하고자 하였다. 제1장에서는 서브유닛 백신 개발에 이용하기 위해 감염 대상의 수 용기와 직접적으로 상호작용하고 침입 이후에는 중화항체를 유도한 다고 알려진 에피토프가 있는 spike단백질의 s1도메인의 아미노산 33번부터 591번까지의 영역에 차후에 경구 백신으로 이용할 수 있 도록 M-cell펩타이드를 결합한 영역(MS1이라 명명한다.)을 E.coli 발현 시스템을 이용해 생산하였으나 불용성 형태의 단백질로 생산 되었다.이와 같은 형태는 생물학적 이용성이 없기 때문에 이를 수 용성 형태로 바꾸기 위해 단백질 수준에서의 가용화 및 재접힘 전 략을 세워 실험하였다.가용화 단계에서는 0,2,4,6,8M의 요소 농 도와 pH 8.5와 pH 12.5를 각각 적용하여 최종적으로는 2M의 요소 농도와 함께 pH 12.5의 환경에서 5mg/ml의 수준으로 불용성 단백 질을 녹였을 때,평균 4.5mg/ml의 가용화 단백질을 얻을 수 있었다. 해당 단계에서 각각 23℃와 37℃에서 생산한 불용성 단백질의 가용 화 정도의 차이가 있는지 보았지만,괄목할만한 차이가 보이지 않았다.다음 단계는 재접힘 과정으로서 가용화된 단백질을 한번에 희석
화 하는 방법(flash dilution method)과 가용화된 단백질을 일정한
속도를 지니게 하여 희석화 하는 방법(pulsatiledilution method)을
비교해보았다.결과적으로 실험에 이용한 단백질에는 한번에 가용화 된 단백질을 넣어주는 방법을 통해 최대 0.21mg/ml의 재접힘된 단 백질을 얻을 수 있었다.제1장의 마지막으로는 이온 교환 크로마토 그래피를 이용한 단백질의 정제이다.본 단계에서는 음이온 교환 수 지를 이용하였고,이를 위해 MS1의 등전점인 5.19보다 높은 pH 범 위를 7.0,7.5,8.0,8.5,9.0으로 각각 설정하여 재접힘된 단백질이 가 장 안정적으로 정제될 수 있는 pH 8.5에서 0.18mg/ml수준의 정제 된 단백질을 얻을 수 있었다. 제2장에서는 재접힘된 단백질의 특성 규명을 위하여 원이색성 분석 과 푸리에변환 적외분광 분석을 도입하였다.제1장에서 진행되었던 재접힘의 과정을 통해서 단백질이 제대로 접혀져 고유의 2차구조를 가지고 있는지 확인해본 결과,원이색성 분석에서는 α나선,β병풍구 조 그리고 불규칙 나사선 구조의 존재가 확인되었다.각각의 서로 다른 성장온도에서의 불용성 단백질로 부터의 구조적 차이는 없는 것으로 밝혀졌으며,성장온도와 상관없이 단백질 2차구조의 종류별 비율에도 큰 차이가 없었다.반면,푸리에변환 적외분광 분석에서는 원이색성분석에서 발견되어졌던 α나선구조와 불규칙 나사선 구조의 존재는 보이지 않았지만 β병풍구조의 존재는 확인되어졌다.또한, 가용화된 단백질내부에서 일부는 다시 응집된 형태를 가지고 있는 것으로 보여졌다.뿐만 아니라 제2장에서는 돼지 유행성 설사병을
수 있는지 그 항원력을 평가하기 위하여 생체 내 면역 실험이 진행 되었다.총 4주 동안 50㎍의 항원이 완전프로인트 항원보강제와 함 께 2주간의 간격을 두고 피하로 주사되어졌다.음성그룹으로는 PBS 가 이용되었고 양성그룹에는 상용 PEDV 경구백신이 이용되어졌다. 혈청의 IgG,IgG1와 Ig2a가 분석되어졌고,세 개의 면역글로불린 모 두 음성,양성 그룹보다 높은 수치를 나타낸 것을 통하여 재접힘 과 정을 통해 불용성 단백질로 생산되어진 MS1이 생물학적 이용성을 가진 수용성 단백질로 재접힘 되었음을 알 수 있다. 모든 결과들을 종합해 보았을 때,불용성 단백질로 생산되어진 돼지 유행성설사병 바이러스의 spike 단백질을 단백질 수준에서 가용화 및 재접힘과정을 통해 약 35%의 수율로 수용성 단백질을 회수할 수 있었다.본 연구를 통해 최적화된 수용성 단백질의 항원 생산 공정 은 불용성 단백질로 생산되어지는 단백질의 수용화 생산 과정에 기 여할 수 있을 것으로 기대된다. 주요어 :가용화,재접힘,내포체 단백질,돼지유행성설사병,서브유 닛 백신,E.coli발현 시스템
