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 temperatureforPED antigenexpression.
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 antigen.
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, insolublefraction.
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 maximum productionofsolubleform wasenhanced.
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, insolublefraction.
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 expressionrespectively.
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 fraction.
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;
S,solublefraction;I,insolublefraction.
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 through,Lane3-4:washingfraction,Lane5-9:eluates.