GST蛋白表达、纯化步骤

ProteinExpressionProtocol:Transformcontrolconstructandtag-fusion-constructsintoE.coliBL21(DE3)competentcell,growovernight;Inoculatesinglecolonyto2~3mlLBmedium,andgrowat37degreefor7~10hoursorovernight;1:100inoculateto3mlLB,bacteriagrowfor2-3hoursat37degree,thenfollow1:2000add1MIPTGtoinduceproteinexpressionovernightat22degree;Spindownbacteriaat12000rpmfor1min,washwith1mlddH2O,add100ul1xPBStoresuspendthedeposition,thensonicate3~5minonice;Spindownat12000rpmfor10min,separatethesupernatantsanddeposition;Boilingwithloadingbufferat100degreefor5min,Spindownat12000rpmfor5min,12%SDS-PAGE;Afterconfirmedproteinexpressedinsupernatants,increasethevolumeofbacteriamedium.1:100inoculateto100mlLB,growbacteriafor2-3hoursat37degree;thenfollow1:2000add1MIPTGtoinduceproteinexpressionovernightat22degree;Spindownbacteriaat8000rpm4degreefor5min,washwith10mlddH2O,add5ml1xPBStoresuspendthedeposition,thensonicate60minonice;Spindownat12000rpm4degreefor10min,separatethesupernatantsanddeposition;Preparethesupernatantsforpurification.蛋白表达注意事项：E.coliBL21比DH5α生长要快，固体培养基上10～12小时即可长斑，液体培养基中8小时后即可生长成较大密度；切勿培养时间过长，防止菌体老化；为了后续实验的便利，应尽量减少包涵体的形成。主要有以下方法可以减少包涵体的形成:降低IPTG的浓度。IPTG终浓度0.2～0.8μM都可以诱导细菌表达蛋白；加入IPTG后，把细菌生长温度降至16～30度。较低的生长温度降低了无活性聚集体形成的速率和疏水相互作用，从而可减少包涵体的形成；添加可促进重组蛋白质可溶性表达的添加剂，培养E.coli时添加高浓度的多醇类、蔗糖或非代谢糖可以阻止分泌到周质的蛋白质聚集反应，在最适浓度范围内添加这些添加剂不会影响细胞的生长、蛋白质的合成或运输，其它添加剂还有乙醇（诱导热休克蛋白的表达）、低分子量的巯基或二硫化合物（影响细胞周质的还原态，从而影响二硫键的形成）和NaCl；供给丰富的培养基，优化培养条件，如供氧、pH等。超声后加入终浓度1%Tritonx-100处理30～60min，有利于去除膜碎片和膜蛋白；若需表达的蛋白含稀有密码子较多，尝试更换E.coli宿主菌株，如Rosseta。若表达毒性蛋白，造成细菌死亡或者生长缓慢，可以使用pLysS菌株。GSTfusionproteinpurification（GlutathioneSepharose4B）Bufferpreparation：Waterandchemicalsusedforbufferpreparationshouldbeofhighpurity.Werecommendfilteringthebuffersbypassingthemthrougha0.45µmfilterbeforeuse.Bindingbuffer:PBS,pH7.3(140mMNaCl,2.7mMKCl,10mMNa2HPO4,1.8mMKH2PO4,pH7.3)Elutionbuffer:50mMTris-HCl,10mMreducedglutathione,pH8.0GST-xPurificationProtocolDeterminethevolumeofGlutathioneSepharose4Brequiredforyourpurification.AsthebindcapacityofGlutathioneSepharose4Bis>5mgglutathione-S-transferase/mlmedium,generally,150ul~200ulslurryfor100mlLBbacterialysateisenough;GentlyshakethebottleofGlutathioneSepharose4Btoresuspendtheslurry;Sedimentthemediumbycentrifugationat12000×gfor1min.Carefullydecantthesupernatant;WashtheGlutathioneSepharose4Bbyadding5slurryvolumesBindingbuffer.Inverttomix;Sedimentthemediumbycentrifugationat12000×gfor1min.Carefullydecantthesupernatant;Repeatsteps3and4twice;Trannsferthemediumtoa15mltube,addthecelllysatetothepreparedGlutathioneSepharose4Bandincubateovernightat4℃.Usegentleagitationsuchasend-over-endrotation;Sedimentthemediumbycentrifugationat6000×gfor5min.Carefullydecantthesupernatantandsaveitformeasuringthebindingefficiencytothemediumi.e.bySDS-PAGE;WashtheGlutathioneSepharose4Bbyadding5slurryvolumesBindingbuffer,transferthemediumtoa1.5mltube.Inverttomix;Sedimentthemediumbycentrifugationat12000×gfor1min.Carefullydecantthesupernatant(=wash)andsaveitforSDS-PAGEanalysis;Repeatsteps9and10twiceforatotalofthreewashes;Elutetheboundproteinbyadding0.5slurryvolumeElutionbuffer.Incubateatroomtemperaturefor1h.Usinggentleagitationsuchasend-over-endrotation;Sedimentthemediumbycentrifugationat12000×gfor1min.Carefullydecantthesupernatant(=elutedprotein);Repeatsteps7and8twiceforatotalofthreeelutions.Checkthethreeeluatesseparatelyforpurifiedproteinandpoolaccordingtotheresults;WashtheGlutathioneSepharose4Bbyadding5slurryvolumesBindingBuffer.Inverttomix;Sedimentthemediumbycentrifugationat12000×gfor1min.Carefullydecantthesupernatant;repeatsteps14and15twice;StoreGlutathoneSepharose4Bat4°Cin20%ethanol.GST融合蛋白纯化注意事项：1.BindingBuffer和ElutionBuffer中加入1–10mMDTT，可以提高蛋白纯度，但是会导致其产率降低；2.GST融合蛋白不与GlutathioneSepharose4B(简称4B)结合：a.超声太剧烈或时间过长会引起蛋白变性，导致蛋白不能与4B结合。需注意设置好超声仪的功率和间隔时间；b.在细胞裂解和加BindingBuffer和ElutionBuffer之前，加入终浓度1–10mMDTT可以显著提高GST融合蛋白的结合效率；c.由于4B在pH值低于6.5或高于8.0结合效率会降低，因此使用前需用pH6.5～8.0的Buffer如PBS进行平衡；d.如果4B已经使用过几次，有必要进行重生或者改用新鲜的4B；3.GST融合蛋白不能有效洗脱：a.洗脱时间不足；b.增大ElutionBuffer的洗脱体积；c.加大ElutionBuffer中还原型glutathione的浓度。Glutathione的推荐浓度是10mM，若此浓度洗脱效果不是很理想可以尝试50mMTris-HCl,20–40mMreducedglutathione,pH8.0的ElutionBuffer；d.增大ElutionBuffer的pH值。ElutionBuffer的pH值调至pH8–9可以提高洗脱效率而不需要增加glutathione的浓度；e.增加ElutionBuffer的离子强度。加入0.1–0.2MNaCl能提高洗脱效率；f.改用新鲜配置的ElutionBuffer；g.ElutionBuffer中加入非离子型变性剂。非特异性的疏水作用可能会阻碍GST融合蛋白从4B上增溶和洗脱。加入0.1%TritonX-100or2%和N-octylglucoside可以显著增加洗脱效率。4.GlutathioneSepharose4B重生。如果GlutathioneSepharose4B上积累了沉淀物，变性的蛋白或者其他非特异性蛋白，导致结合效率降低甚至丧失结合能力。可以用以下方法进行重生：a.去除沉淀物或者变性蛋白：用6M盐酸胍洗2个柱体积后，紧接着用pH7.3的PBS洗5个柱体积；b.去除疏水性的化合物：用70%乙醇洗3～4个柱体积或者用1%Triton™X-100洗2个柱体积后，紧接着用pH7.3的PBS洗5个柱体积。5.最好用新鲜纯化的蛋白用于后续实验，根据实际情况选用蛋白定量试剂盒；6.上述注意事项未提到的问题请查阅说明书。