生物大分子分离纯化原理与技术省公开课一等奖全国示范课微课金奖PPT课件.pptx

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,生物大分子分离纯化,原理与技术,1/115,内容提要,1,、层析原理与技术,2,、凝胶过滤层析,3,、,UNOsphere,离子交换分离技术,4,、,CHT,陶瓷羟基磷灰石分离技术,5,、疏水相互作用与亲和层析,2/115,Life is based on proteins and their interactions,3/115,层析原理与操作,4/115,层析起源和原理,起源,-1906,年，俄国植物学家,Tsweet,原理,-,利用物质分配系数不一样到达分离 目标,原理与操作,Chromatography,层析,色谱,5/115,什么是生物层析,依据生物分子物理化学特征不一样而到达分离,极性：,polarity(solubility,volatility)HIC,RP,离子特征：,ionic characteristics(charge)IEX,大小与形状：,size/mass(diffusion,sedimentation)GF,结构特征与活性位点：,shape(ligand binding,affinity)AC,这些特征区分使不一样蛋白质分子在层析固定相和流动相分配不一样而到达分离,原理与操作,6/115,Gel,Filtration,凝胶过滤,Ion,Exchange,离子交换,Hydrophobic Interaction,疏水层析,反相,Affinity,亲和层析,CHT,羟基磷灰石,原理与操作,7/115,最广泛蛋白质分离纯化方法,条件温和,维持蛋白质空间结构与活性,基于蛋白质对固定相不一样保留到达分离,蛋白质溶于流动相中，经过装填固定相柱子分离,层析原理与操作,8/115,层析,5,个操作步骤,装柱并平衡,上样,平衡,洗脱,再生,上样,装填有层析介质层析柱,分离,分离组分流出,原理与操作,9/115,层析图,原理与操作,第一峰,外水体积,V,o,不与柱中填料相互作用直接从柱中流出。这是样品中未结合物质,V,o,V,e,V,t,蛋白质含量,(A,280,),由,UV,检测器读出，,y,轴,基线,洗脱峰，有些能够很好分离，有些分得不是很好，有部分交叉,有时这里也显示梯度浓度等检测值,时间或体积，,x,轴,10/115,纯化平台硬件结构,层析工作站,层析介质和层析柱,原理与操作,11/115,层析系统,DuoFlow,中高压层析系统,LP,低压层析系统,Profinia,全自动亲和层析系统,手动层析组件,12/115,DuoFlow,中高压层析系统,主要特点：,3500psi,高压力下梯度模式最大流速可达,20ml/min,连续波长，,4,波长同时检测,方形,X,、,Y,轴组分搜集器,软件直观轻易使用,13/115,BioLogic LP,低压层析系统,LP Data View,软件,BioLogic LP,系统,BioFrac,方形组分搜集器,主要特点：,流速准确，,0.01ml/min,检测灵敏度更高,0.001AU,兼容方形搜集器,14/115,Profinia,蛋白质纯化系统,主要特点：,全自动亲和纯化，包含亲和标签、亲和无标签，抗体等纯化,双紫外检测器设计,纯化与脱盐串联一步完成,结果直接汇报目标蛋白浓度和得率,15/115,为何在层析技术中要使用层析仪,介质要求,试验精密度和重现性要求,时间要求,16/115,纯化平台硬件结构,层析介质与层析柱，原理与技术,17/115,层析介质,离子交换层析介质,Unosphere Q,，,S,MacroPrep High Q,High S,DEAE,CM,亲和层析,Profinity IMAC,金属螯合介质,Profinity Epoxide,环氧亲和介质,Affi-Gel Protein A,Affi-Prep Protein A,Affi-Gel,蓝胶，,DEAE,蓝胶，,CM,蓝胶,Affi-Prep,多粘菌素介质,Affi-Gel,硼胶,Affi-Gel,配体固定化活化介质,凝胶过滤层析介质,Bio-Gel P,系列,Bio-Beads S-X,介质,羟基磷灰石介质（,CHT,）,氟代羟基磷灰石介质（,CFT,）,疏水层析介质,Macro-Prep Methyl HIC,Macro-Prep t-Butyl HIC,Bio-Beads SM-2,吸附剂,18/115,层析柱,分析柱,离子交换分析柱：,Uno Q,S,Aminex,分析柱,分析单糖、寡糖、有机酸、有机碱，以及肽和核酸有机小分子,羟基磷灰石分析柱：,Bio-Scale CHT-I,凝胶过滤分析柱：,Bio-Sil,Bio-Silect HPLC,分析柱,反相层析分析柱：,Hi-Pore RP304,Hi-Pore RP318,各种层析介质,Cartridges,用于条件探索,UNOsphere,MacroPrep,CHT,HIC,19/115,层析介质结构原理,Unosphere,MacroPrep,离子交换层析,Q,，,S,，,DEAE,，,CM,疏水层析,-CH3,t-Butyl,亲和层析,Protein A,IDA,Ni,多粘菌素,凝胶过滤,CHT,和,CFT,Unosphere Q,S,MacroPrep High Q,S,MacroPrep DEAE,CM,MacroPrep Methyl HIC,MacroPrep t-Butyl HIC,Profinity IMAC,Profinity Epoxide,Affi-Gel Protein A,Affi-Prep Protein A,Affi-Gel,DEAE,CM,Affi-Prep Polymixin,Affi-Gel,硼胶,Affi-Gel,配体固定化活化,20/115,层析介质及其技术,凝胶过滤层析,离子交换层析,羟基磷灰石层析,疏水层析,亲和层析,21/115,1.Spherical particles packed into a column,2.Sample applied,3.Buffer,mobile phase,and sample move through column,molecules diffuse in and out of matrix,4.large molecules leave the column first followed by smaller molecules in order of size,m,olecules larger than the,matrix,pores,pass straight through.,Diffusion,Buffer,Diffusion into the pores,Diffusion,out of,the pores,Buffer,凝胶过滤,22/115,凝胶过滤层析,分离纯化原理,23/115,A good gel for gel filtration contains about,95%water,凝胶结构,24/115,Steric exclusion leads to early elution,按照分子量大小洗脱,大分子首先洗脱，最小分子在最终面洗脱,25/115,100 1,000 10,000 100,000,Bio-Gel P2,Bio-Gel P4,Bio-Gel P6,Bio-Gel P10,Bio-Gel P30,Bio-Gel P60,Bio-Gel P100,1,800,800,4,000,1,000,6,000,，,用于脱盐,1,500,20,000,2,500,40,000,3,000,60,000,5,000,100,000,100,凝胶过滤层析,凝胶选择,26/115,Bio-Gel P4(800-4,000)for separation digested products from IgG,凝胶过滤层析,凝胶选择,27/115,凝胶过滤层析,Bio-Gel P6(1000-6000),凝胶选择,28/115,凝胶过滤层析,Bio-Gel P10(1500-20,000),凝胶选择,29/115,凝胶过滤层析,Bio-Scale Mini Bio-Gel P6,脱盐预装柱,操作方便，可连接在,DuoFlow,、,LP,、,Profinia,和,Econo,泵上,30/115,Bio-Beads S-X,介质,多孔聚苯乙烯二乙烯微球体,Bio-Beads S-X1,Bio-Beads S-X3,600,Bio-Beads,S-X8,1,000,Bio-Beads,S-X12,400,高分辨率亲脂性分子排阻色谱,2,000,14,000,中草药和天然产物活性成份分离纯化,高分辨率，快速纯化酯类、芳香族，多环、杂环化合物,介质可兼容苯、甲苯、二甲苯、四氯化碳、二甲基甲酰胺、酮、二氯甲烷、二氯代苯、全氯乙烯等有机溶剂,凝胶过滤层析,31/115,1,、三硬脂酸甘油酯，,891,2,、三肉豆蔻酸甘油酯，,729,3,、三月桂酸甘油酯，,645,4,、三辛酸甘油酯，,477,5,、三己酸甘油酯，,393,6,、十六烷，,226,7,、十一烷，,156,Bio-Beads S-X8,分离效果,高分辨率亲脂性分子排阻色谱,Bio-Beads S-X,介质,凝胶过滤层析,32/115,柱长选择,分离：,30,120cm,脱盐：,30cm,以下,洗脱液,离子强度,低,高,离子作用 排阻作用（,0.1-0.5M),疏水作用,pH,中性,流速，,依据层析介质说明，普通很低,样品容量,：,1,3,CV,凝胶过滤层析,操作参数选择,33/115,增加分辨率方法,检测柱效,检测分离度,降低上样体积,降低流速,使用更小介质颗粒介质,连接,2,根,层析柱,34/115,1.,可分离相对分子量从几百到几十万物质,含有,3000,个理论塔板凝胶过滤可将分子量相差,2,倍蛋白质完全分开,6000,个理论塔板可将分子量相差,1.5,倍蛋白质完全分开,;,提议柱高在,80-120cm,直径,=H/30,2.,脱盐,凝胶过滤层析,凝胶过滤层析应用,35/115,凝胶过滤层析,凝胶过滤层析特点：,层析柱规模很大，试验室,1.0-2.530-100cm,，工艺,10,20200cm,，从而设备成本很高；,上样体积少，必须少于柱床体积,3,才能到达很好分离效果，假如大致积样品必须浓缩，从而造成样品损失和增大工艺复杂性；,工艺时间（生产周期）长，甚至,24,小时或以上；,分辨率低；,不需探索纯化条件，按照分子量区带分离,所以，往往用分子筛分离时只适合用于纯化最终一步去热原，或离子,交换上样前脱盐,36/115,cation,weak,strong,anion,weak,strong,DEAE,Diethylamino-ethyl,Q,Quaternary amine,CM,Carboxy-methyl,S,Sulfonate,离子交换层析,离子交换层析类型及其选择,-O-CH,2,CH,2,-NH,+,C,2,H,5,C,2,H,5,-N,+,(CH,3,),3,-O-CH,2,-C-O,-,O,-SO,3,-,37/115,pI,stability range,stability range,与阳离子交换介质结合,+,-,denaturation,denaturation,pH,10,2,离子交换层析,离子交换层析原理,蛋白质滴定曲线,蛋白质净电荷,与阴离子交换介质结合,38/115,Products:,UNOsphere Q&S,Macro-Prep High Q&S,CM,DEAE,AG resins,Equilibration,Sample,Application,Sample,Adsorption,Elution,Regeneration,-,-,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,-,-,-,-,-,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,-,-,-,-,-,-,-,-,-,-,-,-,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,离子交换层析,离子交换层析原理,39/115,sample,application,and wash,elution,equilibration,regeneration,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,-,-,-,-,-,-,-,-,-,-,-,anion,exchanger,bead,离子交换过程,40/115,离子强度,洗脱时多采取离子浓度梯度,常加,NaCl,KCl,LiCl,等,.,在不一样盐浓度（离子强度）及其不一样改变率（梯度）条件下保留行为不一样，需对该条件进行探索。普通，随离子强度增加，蛋白质保留值减小。,缓冲液与,pH,选择适当缓冲体系，起始浓度要尽可能低些,(0.01-0.05M),缓冲液,PH,值：,阳离子低于,pI,一个,pH,单位以上,阴离子通常高于,pI,一个,pH,单位以上,蛋白质在不一样,pH,下保留行为差异较大，所以许对缓冲体系和操作,pH,进行探索，以得到最正确层析条件。,离子交换层析,离子交换层析操作参数选择,41/115,pI,5.5,+,-,pH,10,2,离子交换层析,缓冲液与,pH,pI,7.5,阳离子交换,阴离子交换,碱性蛋白，采取阳离子交换,酸性蛋白，采取阴离子交换,蛋白质净电荷,pH4.0,pH9.0,蛋白质稳定性范围,42/115,pH 7.0,pH 8.0,pH 8.9,pH 6.0,pH6.0,pH7.0,pH 8.0,pH 8.9,缓冲液与,pH,影响,阴离子交换,离子交换层析,离子交换层析操作参数选择,43/115,离子交换层析,离子交换层析操作参数选择,盐溶液梯度影响,44/115,Columns,Media,Bio-Scale Mini High Q Macro-Prep high Q,Bio-Scale Mini High DEAE Macro-Prep DEAE,Bio-Scale Mini High S Macro-Prep high S,Bio-Scale Mini UNOsphere Q Macro-Prep CM,Bio-Scale Mini UNOsphere S Macro-Prep 25 Q,Bio-Scale Mini UNOsphere rapid S Macro-Prep 25 S,UNO Q,UNOsphere Q,UNO S,UNOsphere S,UNOsphere rapid S,离子交换层析,离子交换层析产品,50um,25um,120um,80um,10um,100um,45/115,更高选择性和,分辨率,10%,穿透,载量,:,Unosphere Q:150 mg/ml BSA 600 cm/hr,Unosphere S:30 mg/ml BIgG 600 cm/hr,高流速，低反压,操作压力,:1 week,长久,:Storage in 0.1 M NaOH RT for 1 year,生产效率大大提升,离子交换层析,UNOsphere Q/S,性能参数,46/115,Column 1.1 x 20 cm,Load buffer:20 mM Tris,pH 8.5,Elution Buffer:Load+0.5 NaCl,Load:5 mg/ml BSA,Column 1.1 x 20 cm,Load buffer:20 mM NaAc,pH 5.0,Elution Buffer:Load+0.5 NaCl,Load:1.64 mg/ml hIgG,UNOsphere Q:BSA,UNOsphere S:HIgG,高流速，高载量：,Unosphere,47/115,高流速，低反压：,Unosphere,48/115,Support,Linear velocity(cm/hr),Recovery(%),BSA binding capacity(g/L),Process time(hr),Productivity(g/L/hr),UNOsphere Q,615,100,120,1.58,75.0,Q Sepharose FF,300,99.0,23.0,1.19,19.0,Fractogel EMD TMAE(M),105,99.0,82.0,5.04,16.0,Support,Linear velocity(cm/hr),Recovery(%),Human IgG binding capacity(g/L),Process time(hr),Productivity(g/L/hr),UNOsphere S,1100,98.6,32.0,0.60,53.3,SP Sepharose FF,500,97.8,14.3,0.77,18.5,Fractogel EMD SO3(M),231,97.0,66.4,6.50,10.2,离子交换层析,49/115,UNOsphere,更高生产效率：,更短工艺时间,短时间内处理大量体积样品,更小层析柱，更少介质,更高产量,更短生产周期,生产成本下降,离子交换层析,蛋白质纯化，尤其是从原材料或发酵中间体目标蛋白大量捕捉,中间纯化,UNOsphere Q/S,应用,50/115,4 Process Steps,1.Polymerization,2.Washing,3.Deagg/Sizing,4.Base Treatment,Monomer,Cross-linker,Ionomer,Surfactant,Salt,Solvent,Initiator,Heat,Base,Treatment,Bottling,IPA Wash,Unsized UNOsphere Bead,Wet Sizing,Sized UNOsphere Bead,Deagg,UNOsphere,制造过程,51/115,UNOsphere,%cross-linker:S25%,Q25%,离子交换层析,UNOsphere Q/S,结构,52/115,Continuous network of polymer nodules,0.5-1 m,Channels,3-5 m,Q,：,(CH,3,),3,N,+,S,：,SO,3,-,Profinity IMAC,：,IDA-Ni,，,Cu,，,Co,离子交换层析,UNOsphere Q/S,结构,53/115,Reference:T.Ogawa et.al.Hydroxyapatite Conference,陶瓷羟基磷灰石（,CHT,）分离技术,合成,54/115,左边,:Bio-Gel HT/HTP,羟基磷灰石,右边,:CFT/CHT,TM,陶瓷羟基磷灰石,I&II,晶体与陶瓷结构,陶瓷羟基磷灰石（,CHT,）分离技术,55/115,Ca,10,(PO,4,),6,(OH),2,5,个带正电荷,Ca,离子对（,C,位点）,2,个磷酸三价离子，每个含有带,6,个负电荷氧原子（,P,位点）,2,个羟基,于其它层析介质不一样，,CHT,骨架是化学反应表面,CHT,陶瓷羟基磷灰石晶体结构,陶瓷羟基磷灰石（,CHT,）分离技术,56/115,产品类型与参数,陶瓷羟基磷灰石（,CHT,）分离技术,57/115,蛋白质带正电氨基,经典阳离子交换,用中性盐溶液,(NaCl),或缓冲盐溶液,(PO4),洗脱,初级保留机制,陶瓷羟基磷灰石（,CHT,）分离技术,58/115,带负电羧基,经典金属螯合作用，并受离子排阻调整,比离子间静电相互作用强,1560,倍,在无,PO4,条件下不能被任何浓度,NaCl,洗脱,用,PO4,洗脱,初级保留机制,陶瓷羟基磷灰石（,CHT,）分离技术,59/115,大部分大分子蛋白质以两种保留机制联合模式结合：,Ca,亲和与阳离子交换,酸性蛋白质结合，如白蛋白（,albumin,）以钙亲和作用为主，阳离子交换仅有轻微作用，这意味着,NaCl,对白蛋白载量和保留影响是非常有限。,碱性蛋白质，如,IgG,以阳离子交换作用为主，其载量和保留受,NaCl,影响显著,注：,对于带负电荷酸性蛋白质，不论样品是否含有,NaCl,，都对上样时吸附载量和保留无影响，这意味着能够将捕捉阶段取得中间体样品无需脱盐处理即可上样到,CHT,上进行高分辨率中间纯化,混合保留机制,陶瓷羟基磷灰石（,CHT,）分离技术,60/115,Equlibrate:5mM NaPO4 pH 6.5,Gradient:5mM300mM NaPO4,Eq:5mM PO4,0.3M NaCl pH 6.5,Grad:5mM300mM PO4,0.3M NaCl,IgG MAb,BSA,混合保留机制,陶瓷羟基磷灰石（,CHT,）分离技术,61/115,单克隆抗体，多克隆抗体纯化,重组疫苗,抗体片段,重组蛋白质,酶,核酸,:DNA/RNA(,单双链,),膜蛋白质,羟基磷灰石应用,陶瓷羟基磷灰石（,CHT,）分离技术,62/115,优点,独特分离机制,高再生能力,高分辨率,轻易规模放大,轻易进行方法开发,高机械稳定性,高化学稳定性,层析柱寿命长,易于将,HT,工艺直接转移到,CHT/CFT,上,CFT,比,CHT,更耐酸,颗粒大小：,20um,、,40um,和,80um,陶瓷羟基磷灰石（,CHT,）分离技术,63/115,Type I,因为含有更高表面积，,I,型含有更高蛋白载量，而且含有更大保留,.,Type II,因为含有大孔结构，,II,型含有更高核酸载量，能分离单链和双链,DNA,，和超螺旋与非超螺旋,DNA,白蛋白不能与,II,型结合，对于一些含白蛋白抗体样品，,II,型分离纯化更有利,.,大分子重组疫苗中间纯化和大规模制备,怎样选择类型,陶瓷羟基磷灰石（,CHT,）分离技术,64/115,CHT,纯化或去除,DNA,机理,陶瓷羟基磷灰石（,CHT,）分离技术,65/115,分离纯化原理与操作参数选择,羟基磷灰石（,CHT,）,影响蛋白质色谱行为操作参数：,CHT,类型,NaCl,浓度,PB,缓冲液浓度梯度,双梯度洗脱,PB,缓冲液梯度含不一样,NaCl,浓度,缓冲液,pH,值,双梯度洗脱模型,先以,0-500mM NaCl,，再以,0-500mM,磷酸钾,66/115,不一样类型,CHT,陶瓷羟基磷灰石在不一样,pH,下不一样蛋白质保留时间（,min,）,羟基磷灰石（,CHT,）,67/115,不一样蛋白质分离纯化,Protocol,：,IgG,羟基磷灰石（,CHT,）,68/115,不一样蛋白质分离纯化,Protocol,：,球形蛋白质，质粒,羟基磷灰石（,CHT,）,69/115,不一样蛋白质分离纯化,Protocol,：,酸性蛋白质,羟基磷灰石（,CHT,）,70/115,双梯度洗脱,羟基磷灰石（,CHT,）,0-0.5M,NaCl,0-0.4M,NaPO,4,玉米种子中纯化转基因抗体,IgG,71/115,图,1 CHT-I,柱纯化,1-AT,。柱尺寸：,752mm(10ml),；上样量：,5.0ml,；,Buffer A,：,10mM,磷酸钠缓冲液（,pH7.0,）；,Buffer B,：,400mM,磷酸钠缓冲液（,pH7.0,）；梯度：,0-5%B,和,5-30%B,各,5,个柱体积，,30-100%B 10,个柱体积；流速：,2.5 ml/min,。分部搜集组分为,1.5ml,，搜集以下：搜集组分,I-VIII,分别为：运行组分（图上方轴）,12-17,、,23-24,、,42-49,、,49-50,、,51-53,、,54-58,、,69-71,和,74-77,。绿条框指示为,1-AT,洗脱液。,CHT,纯化转基因羊奶中重组人,1-,抗胰蛋白酶,羟基磷灰石（,CHT,）,PI,其中，,1-AT,，,pI,5.27,乳白蛋白，,pI,5.16,-,乳球蛋白,pI=4.77,5.27,4.77,5.16,72/115,Market trend of therapeutic antibodies,Twenty approved antibody therapeutics currently available for the treatment of various diseases-Reopro,Rituxan,Herceptin,Remicade,Synagis,Avastin,Erbitux,Xolair,Raptiva,Lucentis,Tysabri,etc.,The antibody therapeutics market is expected to grow by about 30%annually reaching excess of$22 billion by.,Reference:,Antibody Therapeutics:Protein Development,Market Trends,and Strategic Issues,Revised edition of Monoclonals,Fermentation,Harvest,Recovery,Downstream,Processing,Bulk Drug,Substance,74/115,Monoclonals Downstream Process A Generic Approach,75/115,Critical contaminants in downstream process development,Contaminants,Molecular weights,Isoelectric points,IgG Aggregates,300,000,Same as IgG,Protein A,42,000,4.9-5.1,DNA,1,000-1,000,000,Highly acidic,Endotoxin,100,000,Highly acidic,Host cell proteins,Vary,Various,Viruses,1,000,000,3,3,2,2,89/115,Retention of endotoxins,Endotoxins are highly acidic due to a high content of,phosphoryl and carboxyl,residues.,Both should have strong affinity for CHT calcium.Elution in the absence of phosphate should not occur.,Some endotoxins have,amino groups,which may cation exchange with CHT phosphates at low ionic strength.,Endotoxins occur in a range of,aggregation,states,into the millions of Daltons.Most of the charges are,internalized,because of their association with the hydrophobic lipid A region.Both size and charge shielding may affect retention.,Endotoxins are frequently,complexed with proteins,and other cell wall components that may also influence retention,90/115,IgG zone,IgG zone,NaCl 0-1.5M,Clean with 0.5M PO4,PO4 0-0.3M,Clean with 0.5M PO4,Affinity of endotoxins to CHT,LPS mixture from,E.Coli,Salmonella and Pseudomonas,CHT type I,40 micron,300 cm/hr,91/115,Removal of host cell proteins(HCP),HCP are unique for each cell line.,Hundreds to thousands of HCPs derived from cell or cell debris are present in fermentor fluid.,Bulk of HCPs is removed across protein A chromatography.,Mixed mode interaction with CHT can offer unique opportunity for removal of residual HCPs.,92/115,The effect of NaCl and phosphate on CHOP clearance,CHO protein(ppm),Protein A eluate,58.3,NaCl gradient pool,40 1,Protein A,ng -162 6,DNA,ng 9.9x10,5,3.8x10,4,12,Endotoxin,EU 2.6x10,3,5.0 x10,2,3 logs reduction by PCR,reduced to Cl,-,Br,-,NO,3,-,ClO,4,-,I,-,SCN,-,Cations:Mg,2+,Li,+,Na,+,K,+,NH,4,+,蛋白质以高浓度盐溶液结合与疏水层析柱上，以低浓度盐溶液洗脱,原理,疏水层析,MacroPrep Methyl HIC,MacroPrep t-Butyl HIC,产品,100/115,疏水层析,生物分子表面大都有或强或弱疏水区域，在不一样环境下，与各种疏水介质产生不一样强弱结合,高离子强度可加强疏水性,跟离子交换相反，高盐吸附，低盐洗脱；洗脱样品又 可直接或稍加稀释后加上其它层析柱，作为连接层析 步骤桥梁，取代盐析沉淀技术,比反相层析配体密度低很多，无须有机溶剂洗脱，保留生物活性,配体种类繁多，极难预测哪一个、哪一个条件最适合，可用疏水层析试盒选择介质,101/115,作用原理,溶质,曝露在外疏水基,配基,水分子,大量水分子,D,G,=,D,H,-,T,D,S,102/115,为何使用疏水层析,?,离子交换技术、羟基磷灰石、凝胶过滤技术、亲和层析技术补充,温和，非变性条件纯化,互补选择性,高收率,浓缩技术,103/115,操作参数选择,疏水层析,疏水介质选择：甲基，丁基，苯基疏水,疏水性强蛋白质：甲基，丁基,疏水性弱蛋白质：丁基，苯基,盐：硫酸铵，醋酸铵等等,缓冲体系和,pH,洗脱梯度,104/115,Sample:GFP sample brought up to 2 M AS,filtered,Load:5 ml,Buffer:A:2 M(NH,4,),2,SO,4,+0.1 M NaP,pH 7,B:0.1 M NaP,pH 7,Flow Rate:3 ml/min(230 cm/hr),Macro-Prep,甲基疏水,苯基疏水,20%EtOH,疏水层析,105/115,Profinity IMAC,纯化,His-tagged Protein,Chelex 100,纯化,DNA,，,PCR,样品制备,Affi-Gel Protein A,纯化单克隆抗体,Dye-Affi-Gel Blue(DEAE or CM),纯化单抗，分离,HSA,Affi-Gel,肝素凝胶,各种蛋白，如凝结因子、蛋白酶、核酸酶、脂酶,Affi-Gel Polymixin,去除内毒素（热原）,Affi-Gel,硼胶,分离核苷酸、核苷、儿茶酚胺、糖类等小分子,亲和层析,产品与应用,106/115,Equilibration,Sample application,Binding and washing,Desorption and elution,Equilibrate the column and the sample to binding conditions.,Matrix,Specific ligand,亲和层析,107/115,亲和层析,Equilibration,Sample application,Binding and washing,Desorption and elution,Target binds,Others wash out,Apply sample under binding conditions.,108/115,亲和层析,Equilibration,Sample application,Binding and washing,Desorption and elution,Target elutes,Change the eluent to elute the target.,109/115,Profinity IMAC Resins,固定化金属螯合层析：,Immobilized Metal Affinity Chromatography,（,IMAC,）,基于纯化重组组氨酸标识蛋白设计，专门纯化,His-tagged Protein,螯合带电离子，如,Zn2+,Ni2+,Cu2+,现有产品为螯合,Ni2+,介质,UNOsphere,技术,更加好流动特征，在高流速下载量，目标蛋白得率和纯度不会受到影响。,IDA,110/115,各种流速下载量和分辨率,高流速，高载量，高分辨率，低反压,Profinity IMAC Resins,高流速、低反压,111/115,不一样,IMAC,介质纯化氨基肽酶,(aminopeptidase),纯度比较,Profinity IMAC,IDA ligand,Profinity IMAC Resins,112/115,使用,DuoFlow,系统进行,Profinity IMAC,循环性能研究,Strip 50mM EDTA,50mM sodium phosphate,300mM NaCl(pH7.5),Clean 50mM sodium acetate,300mM NaCl(pH4.0),Charge 100mM NiSO4(pH4.0),Clean 50mM sodium acetate,300mM NaCl(pH4.0),Equilibrate 50mM sodium phosphate,300mM NaCl(pH8.0),Sanitize 1.0N NaOH,Sample E,.coli,.lysate containing 3.51mg of 75kD r-His-tagged protein,1mL Profinity IMAC,柱,201,次循环试验,Profinity IMAC Resins,113/115,Profinity IMAC Resins,纯