蛋白纯化策略.pdf

1/GE/Purification Strategies纯化策略纯化策略Introduction 介绍Before Purification 纯化前的准备Strategies 纯化策略Purification of recombinant and native Protein 纯化实例How to get desired resolution?如何取得预期的结果？Summary总结ContentGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.2/GE/Introduction 介绍Before Purification 纯化前的准备Strategies 纯化策略Purification of recombinant and native Protein 纯化实例How to get desired resolution?如何取得预期的结果？Summary总结Content4/GE/2010-4-13Principles of operation for chromatography techniques 层析原理Gel Filtration 分子筛Ion Exchange 离子交换Hydrophobic interaction疏水层析Affinity亲和Reversed phase反相SOOOGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.3/GE/5/GE/2010-4-13How to use these separation technologies to perform a SUCCESSFULpurification?如何运用各种分离纯化技术来实现成功成功的分离纯化？QuestionIntroduction 介绍Before Purification 纯化前的准备Strategies 纯化策略Purification of recombinant and native Protein 纯化实例How to get desired resolution?如何取得预期的结果？Summary总结ContentGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.4/GE/7/GE/2010-4-13目标:开发有效的蛋白质分离纯化工艺 Maintained biological activity 活性 Sufficient purity and quantity 纯度&规模 Good economy 经济性8/GE/2010-4-13pg ng g mg g kg治疗用蛋白治疗用蛋白结构研究结构研究功能研究功能研究质谱质谱设定目标：需要的蛋白量用于免疫的抗原用于免疫的抗原Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.5/GE/9/GE/2010-4-13Extremely high 极高High 高Moderate 中等 therapy治疗用 in vivo studiesPK/PD 动物实验 crystallization for x-ray studies 结晶 N-terminal sequencing of an unknown protein N端测序 most physical-chemical characterization methods 鉴定 antigen for monoclonal antibody production免疫终产品纯度要求 一般原则一般原则:10/GE/2010-4-13选择不同规模、不同功能的系统KTAprime plusKTAexplorerKTAprocessKTApilotKTAxpressLAB SCALEBIOPROCESS SCALESIMPLE PURIFICATIONADVANCED PURIFICATIONINCREASED THROUGHPUTLAB SCALELOW THROUGHPUTKTApurifierGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.95%及以上6/GE/11/GE/2010-4-13开始纯化之前样品来源 原核 or 真核?胞内 or 胞外?细胞破碎?活性,复性?目标蛋白的性质 等电点 分子量 稳定性(pH/salt,etc)Inner membranePeptidoglycanOuter membraneLipopolysaccharide70 70 70 210 胞内胞内2000 proteins100 proteins细胞间质细胞间质10 proteins胞外胞外12/GE/2010-4-13Target protein stability window目标蛋白的稳定性Determination of a suitable ammonium sulfate concentration and pH screening range for HICGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.microcal 差示扫描量热仪7/GE/13/GE/2010-4-13开始纯化之前建立目标蛋白特异性检测方法 Activity Assay(specific)快速可靠的活性检测方法A rapid and reliable assay for the target protein Purity determination 纯度测定方法 e.g.SDS-PAGE电泳,HPLC高效液相层析 Total protein determination 总蛋白测定 e.g.colorimetric method 比色法主要杂质的性质及检测方法AgIntroduction 介绍Before Purification 纯化前的准备Strategies 纯化策略Purification of recombinant and native Protein 纯化实例How to get desired resolution?如何取得预期的结果？Summary总结ContentGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.8/GE/15/GE/2010-4-13策略1：纯化三步曲(CIPP)Three phase strategy puritystepcaptureintermediatepurificationpolishingisolate product,concentrate,stabilizeremove bulkimpuritiesachieve final purity,remove trace impurities,structural variants,aggregates etc.粗纯中度纯化精纯粗纯中度纯化精纯16/GE/2010-4-13分辨率速度收率载量Capture 粗纯Initial purification of thetarget molecule fromcrude or clarified sourceMaterial 样品为粗料液Concentration andstabilization 浓缩，稳定蛋白-e.g.removal of proteases 去除蛋白酶Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.9/GE/17/GE/2010-4-13Intermediate purification中度纯化Removal of bulk impurities去除主要杂质分辨率载量速度收率18/GE/2010-4-13分辨率分辨率Polishing 精纯Final removal of trace contaminants,e.g.structural variants of the target protein最终去除痕量杂质，如目标蛋白的结构变体等载量速度收率Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.10/GE/19/GE/2010-4-13Three phase strategy 三步纯化策略 Ranking of chromatography techniques粗纯中度纯化精纯粗纯中度纯化精纯离子交换疏水亲和分子筛反相离子交换疏水亲和分子筛反相20/GE/2010-4-13MiniBeads3 mmicro-purificationMonoBeads10 mpolishingSOURCE1515 mpolishingSOURCE 3030 mintermediate Impact of bead sizes 粒径的影响SepharoseHP 34 mintermediate Sepharose FF 90 mcapture Capto90 m capture 分辨率反压分辨率反压Superdex pg 34 mpolishing Superdex13 mpolishing90 m30 m15 m10 mGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.11/GE/21/GE/2010-4-13策略2：步骤越少,收率越高Yields from multi-step protein purifications纯化步骤纯化步骤100806040200%process yield5 10 1595%yield/step90%yield/step85%yield/step70%yield/stepMinimize loss of material in each stepReduce number of steps22/GE/2010-4-13纯化表一种膜结合蛋白,白细胞三烯 C4 合成酶,18 kDaprotein宿主:Pichia pastoris 毕赤酵母32.626.870.4291100活性收率%9733672.761纯化倍数fold28351.11.141脱盐/浓缩9.6292.90.1419亲和 219.5763.90.1330亲和 10.83143931.74226溶解0.291083741.7220匀浆比活mol/mg/min总活性mol总蛋白量mg蛋白浓度mg/ml体积ml纯化步骤纯化步骤Wetterholm et.al.,Prot Expr.Purif.60(2008)1-611.19.224.2100Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.12/GE/23/GE/2010-4-13策略3：交替运用互补技术,合理衔接-将分离机理互补的技术进行组合,交替运用不同的层析方法(e.g.IEX,HIC and SEC)-减少不同层析技术间的样品处理(e.g.浓缩,置换缓冲液)-尽量简单24/GE/2010-4-13低盐高盐IEX综合考虑起始和结束条件进行衔接增加特定条件特定洗脱条件AC高盐低盐HIC不结合降低常液洗脱洗脱条件洗脱条件结合条件结合条件050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120mlGF技术技术浓度浓度增加增加Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.13/GE/25/GE/2010-4-13设计合理的纯化路线Combine techniques 使用不同层析技术使用不同层析技术complementary selectivities 选择性互补minimized sample handling 尽量避免样品处理步骤捕获捕获精细纯化精细纯化IEXHICGFHICGFIEX(NH4)2SO4中度纯化中度纯化GFGFACIEXACAC26/GE/策略4：线性放大固定以下条件：固定以下条件：1.相同填料2.相同缓冲液3.相同线性流速相同线性流速/相同柱高相同柱高4.相同样品浓度和缓冲条件5.相同样品体积和柱床体积比例6.相同梯度体积和柱床体积比例放大：放大：1.柱直径2.体积流速3.上样体积ml/min x 60cm2线性流速线性流速:cm/h=cm x 60cm/hr停留时间停留时间:min =Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.14/GE/27/GE/2010-4-13Scale-up:从实验室到工业生产规模KTApilot卫生级中试层析系统卫生级中试层析系统KTAprocess大规模生产层析系统大规模生产层析系统KTAexplorer快速全自动工艺开拓系统快速全自动工艺开拓系统28/GE/2010-4-13Scale-up:从实验室到工业生产规模2.01.000123456(column volumes)A280 nm2.01.000123456(column volumes)A280 nmFineLINE 200LFineLINEFineLINE 200L200L400-倍放大倍放大400400-倍放大倍放大倍放大倍放大10 mm 直径柱直径柱10 10 mm mm 直径柱直径柱直径柱直径柱KTA explorer10KTA process23.6-ml 柱9.4-litre 柱同一KTA平台上的直接线性放大Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.15/GE/Introduction 介绍Before Purification 纯化前的准备Strategies 纯化策略Purification of recombinant and native Protein 纯化实例How to get desired resolution?如何取得预期的结果？Summary总结Content30/GE/2010-4-13Tagged protein标签蛋白Non-tagged protein非标签蛋白Native andrecombinantProteins天然和重组表达的蛋白Simple PurificationAffinity+Gel filtrationHigh purityMulti-Step PurificationCIPPCapture Intermediate PurificationPolishingProtein purification strategies蛋白纯化策略Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.16/GE/31/GE/2010-4-13DAOCS-纯化策略去乙酰氧化头孢菌素去乙酰氧化头孢菌素 C 合成酶合成酶纯化目标DAOCS性质准备样品精细纯化精细纯化粗纯粗纯快速探索填料(scouting)快速探索梯度(scouting)中度纯化中度纯化快速探索填料(scouting)快速探索梯度(scouting)得到得到 5-10 mg DAOCS，纯度足够进行结晶和，纯度足够进行结晶和X-晶体衍射分析一个工作日内完成整个纯化流程晶体衍射分析一个工作日内完成整个纯化流程32/GE/2010-4-13DAOCS 目标蛋白的性质参数参数等电点分子量盐稳定性一般稳定性对纯化设计的影响对纯化设计的影响在粗纯时使用中性 pH 进行阴离子交换层析用 Superdex 75 prep grade 凝胶过滤技术进行精细纯化(3-70kD)可以使用疏水层析在缓冲液中加 DTT 工艺设计重点缩短整体纯化时间数值数值4.834 5002 M(NH4)2SO4容易氧化Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.17/GE/33/GE/2010-4-13DAOCS 注意事项在所有缓冲液中加 DTT 使所有半胱氨酸残基保持还原状态,防止氧化加入蛋白酶抑制剂以防止蛋白酶解用 SDS-PAGE 检查每一个组份中 DAOCS终产品通过酶活测定来确认生物活性34/GE/2010-4-13样品预处理细菌菌体重悬于裂解缓冲液中超声破碎菌体沉淀DNA(optional)20,000 xg离心澄清来源:从 S.Clavuligerus 克隆得到的DAOCS 基因 和在大肠杆菌的胞浆中扩增Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.18/GE/35/GE/2010-4-13选择粗纯的层析技术pI=4.8,阴离子交换阴离子交换:-快速,浓缩-样品处理简单分离机理:带电性质差异考虑到DACOS蛋白的酸性 pI 和稳定性,缓冲液选择中性 pH36/GE/2010-4-13pH的选择(关键参数)Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.19/GE/37/GE/2010-4-13KTA 自动缓冲液制备 BufferPrepBuffer is titrated on pump APump B controls salt conc.38/GE/2010-4-13粗纯-在 KTA上优化梯度线性梯度阶段梯度优化梯度线性梯度阶段梯度优化梯度阴离子交换层析柱:HiPrep 16/10 Q XL0102030min01002003001002003004000020406080mAUmlmS/cm01002001000200030000020406080mAUmlmS/cm01020min01002001000200030000020406080mAUmlmS/cm01020minGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.20/GE/39/GE/2010-4-13选择中度纯化技术疏水层析HIC:-可以跟 IEX 互补衔接,减少样品处理步骤(只需加盐)-DAOCS 在高盐下能保持稳定分离机理:疏水性差异蛋白质疏水性质很难预测:筛选适合填料40/GE/Columns:RESOURCEISO 异丙基RESOURCE ETH 醚基RESOURCE PHE 苯基Sample vol:10 mlBuffer A:2 M ammonium sulfate,50 mM Tris-HCl,1 mM EDTA,1 mM DTT,pH7.5Buffer B:buffer A without ammonium sulfateFlow rate:3 ml/minSystem:KTAFPLC中度纯化 疏水填料筛选Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.21/GE/41/GE/2010-4-13KTA crystal系统(自动多维纯化)42/GE/2010-4-13选择精细纯化层析技术分子筛 SEC:-简单-有力的补充IEX和HIC分离机理:分子量差异最适合分离二聚体,寡聚体和聚合物Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.22/GE/43/GE/2010-4-13DAOCS 纯化-结果粗提粗提中度纯化中度纯化精细纯化精细纯化08060402010020040060080010000mlmAU01002001000200030000020406080mAUmlmS/cm01002001002003004000mlmAuHiPrep 16/10 Q XLSOURCE 15 ISO,内径内径 16 mm,长度长度 50 mmHiLoad 16/60 Superdex 75 prep grade44/GE/2010-4-13中度纯化中度纯化DAOCS 纯化-总结工艺优化工艺优化快速探索填料(scouting)快速探索梯度(scouting)快速探索填料(scouting)快速探索梯度(scouting)优化好的纯化流程优化好的纯化流程样品预处理样品预处理HiPrep 16/10 Q XLSOURCE 15ISOHiLoad 16/60 Superdex 75 prep grade粗纯粗纯精细纯化精细纯化浓缩浓缩60 分钟分钟30分钟分钟40分钟分钟120分钟分钟80分钟分钟时间时间Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.23/GE/45/GE/2010-4-13层析技术的衔接Most common粗纯粗纯精纯精纯IEXHICGFHICGFIEX(NH4)2SO4中度纯化中度纯化GFGFACIEXACACGeneric purification strategy!通用的工艺路线！通用的工艺路线！46/GE/2010-4-13 83 mg homogenous proteinobtainedY.-F.Liao et al.(1996)J.Biol.Chem.271,28348-28358 Capture with step gradient;730 mg of total protein applied63622719UFGFAIEXHICSuperdex 200prep gradeQ SepharoseFast FlowPhenyl Sepharose High PerformanceUltrafiltrationTechniquePurificationfactorComment从毕赤酵母纯化重组的a-甘露糖苷酶Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.24/GE/47/GE/2010-4-13重组人白蛋白的下游纯化工艺Transport offrozen CCS from NCPC Storage of the CCSThawing ofthe CCS Filtration 1pH adjustment 1Heat treatment 1pH adjustment 2Filtration 2Multimodal cation exchangerCapto MMC(salt tolerant)pH adjustment 3Heat treatment 2Ultra/dia-filtrationFiltration 3Final productAminobutylSepharose FFFiltration 4Filling in bottlesPhenyl Sepharose Fast Flow(High Sub)Filtration 548/GE/2010-4-13层析技术的衔接Hydrophobic&membrane proteins捕获捕获精细纯化精细纯化IEXHICGFHICGFIEX(NH4)2SO4中度纯化中度纯化GFGFACIEXACACGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.25/GE/49/GE/2010-4-13硫酸铵沉淀疏水非结合模式阴离子阳离子亲和阳离子亲和720240818647 缓冲液中加入蛋白酶抑制剂 4度下纯化 杂质结合 洗脱条件作为下一步骤结合条件 得到10 mg 均一蛋白A.Tobin et al.(1996)J.Biol.Chem.271,3907-3916HiTrapHeparin HP技术纯化倍数猪脑匀浆ammonium sulfateprecipitationButyl SepharoseFast FlowRESOURCEQRESOURCE SCommentG protein receptor kinase purificationG蛋白受体激酶的纯化50/GE/2010-4-13E.coli cellsInclusion bodies 包含体Washed pellet 沉淀清洗Renatured rhGM-CSF 复性Phenyl Sepharose6 Fast Flow(high sub)Q Sepharose Fast FlowSuperdex75 prep gradeHICAIEXGF溶解(6 M GuHCl)稀释复性Purification of rhGM-CSF(MW14.6k,pI 5.4)重组人粒细胞-巨噬细胞集落刺激因子Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.26/GE/51/GE/2010-4-13rhGM-CSF的纯化Step 1.Phenyl Sepharose6Fast Flow(high sub)Step 1.Phenyl Sepharose6Fast Flow(high sub)Step 3.Superdex75 prep gradeStep 3.Superdex75 prep gradeStart 4344490 1828824 -781920-1.HIC 880220 213840 8.6405 19312.AIEX 620 88 1556201.4874.663.GF 1045 62 9405 16.5841.04Start 4344490 1828824 -781920-1.HIC 880220 213840 8.6405 19312.AIEX 620 88 1556201.4874.663.GF 1045 62 9405 16.5841.04StepTotal vol mlTotal protein mgTotal Endotoxin EUTotal DNA ngEndotoxinClearanceDNA ClearanceSpecific activity of final product:ca.3.3 x 10e7 units/mg,Specific activity of final product:ca.3.3 x 10e7 units/mg,52/GE/2010-4-13Tagged protein标签蛋白Non-tagged protein非标签蛋白Native andrecombinantProteins天然和重组表达蛋白Simple PurificationAffinity+Gel filtrationHigh purityMulti-Step PurificationCIPPCapture Intermediate PurificationPolishingProtein purification strategies蛋白纯化策略Generated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.27/GE/53/GE/2010-4-13带标签的融合表达的重组蛋白优势：简单的亲和纯化检测方便可以进行柱上复性产率提高(expression/stability)溶解度提高/folding劣势：可能需要去除标签标签可能干扰结构或功能54/GE/2010-4-13不同的标签Histidine+Small+Cleavage often not needed+Many references+Cheap-Inclusion bodies-Non-specific purificationMBP+Increases solubility&yield+Specific purification-Large-Cleavage often necessaryStreptag II+Small+Cleavage often not needed+Very specific purification-Expensive-Not many references yetGST+Increases solubility&yield+Specific purification-Large-Cleavage often necessary-Binding capacityGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.28/GE/55/GE/2010-4-13标签蛋白的纯化趋势Affinity+gel filtration Double tagging affinity+affinity+gel filtration56/GE/2010-4-13层析技术的衔接Proteins with known affinity ligand-tagged proteins,antibodies捕获捕获精细纯化精细纯化IEXHICGFHICGFIEX(NH4)2SO4中度纯化中度纯化GFGFACIEXACACGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.29/GE/57/GE/2010-4-13Affinity+gel filtrationAutomated two-step purification on KTAxpressMBPTrap HP HiLoad 16/60 Superdex 200 pg58/GE/2010-4-13双标签得到全长蛋白Protein:Strep tag II-Protein X-(His)6Mr 15 000Sample:15 ml E.coli lysateSystem:KTAxpressAcknowledgement:Martina Nilsson,Biovitrum,Stockholm,SwedenRun 1Run 3Run 2FT1FT3FT2E1E2E3Run 1Run 3Run 2FT1FT3FT2E1E2E3For two AC steps in series,check compatibility of start and end buffer conditionse.g.,Strep tag II tagged protein binds to StrepTactin Sepharosein presence of imidazoleHisTrap HP 1 mlStrepTrap HP 1 mlHisTrap HP 1 ml+StrepTrap HP 1 mlProtein X(His)6Streptag IIGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.30/GE/59/GE/2010-4-13需要精细纯化步骤Size heterogeneitiesAggregationProteolytic fragmentationuse GFuse IEXCharge heterogeneitiesHeterogeniety in N-or C-terminusPost-translational modifications e.g.phosphorylation,glycosylation etc.Introduction 介绍Before Purification 纯化前的准备Strategies 纯化策略Purification of recombinant and native Protein 纯化实例How to get desired resolution?如何取得预期的结果？Summary总结ContentGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.31/GE/61/GE/How to get desired resolution?V2-V1(W1+W2)/2Rs=Selectivity or Efficiency?选择性 or 柱效?62/GE/2010-4-13Selectivity 选择性“Right“technologies and parameters to maximize the differences between proteins!Separation technologies 分离技术 Chromatography media screen 层析填料筛选 Separation conditions screen 分离条件筛选 lower selectivityhigh selectivityGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.32/GE/63/GE/2010-4-13Separation technologies 分离技术GelfiltrationIonexchangeHydrophobic interactionAffinity Reversed phaseSOOO64/GE/2010-4-13Separation media screen 层析填料筛选1.SuperdexPeptide2.Superdex 753.Superdex 2004.SephacrylS-100 HR5.Sephacryl S-200 HR6.Sephacryl S-300 HR7.Sephacryl S-400 HRKav1.0-0.8-0.6-0.4-0.2-1000 10,000 100,000 1,000,000 10,000,0001234 567Superdex 75分离范围:3,00070,000 DaSuperdex 200分离范围:10,000600,000 DaGenerated by Foxit PDF Creator Foxit Softwarehttp:/ For evaluation only.33/GE/65/GE/2010-4-13Separation conditions screen分离条件筛选pH 3pH 10anion exchangercation exchangercharge on protein-+pH:关键参数66/GE/2010-4-13Efficiency 柱效A powerful tool to improve Resolution!“Smaller”media v.s.back-pressure and scale-up Uniform media in size distr