层析分离纯化技术蛋白质纯化策略简易纯化选择GST 融合蛋白的纯化任何规模纯化都比较简单GST MicroSpin Purification Module 立即可用, 50 预装柱含缓冲液和所需试剂可纯化每柱400 μg, 体积达400 μl 样品GSTrap 柱20 分钟内每1 ml 柱12 mg 或每5 ml 柱60 mg 使用针筒，蠕动泵，或者KTA 系统添加剂兼容GSTrap GSTrap Glutathione Sepharose Fast Flow 适合装填在高效层析柱中和层析系统结合使用，并用作放大GST 融合蛋白的检测GST 融合蛋白的检测GST 96- 孔板ELISA 检测试剂盒快速酶测定每块板可检测50 样品适合筛选表达系统和层析组份( His)6 融合蛋白的纯化和检测任何规模纯化都比较简单His MicroSpin 纯化模组立即可用, 50 预装柱连缓冲液和试剂每柱可纯化达100 μg, 体积达400 μl 样品HisTrap Kit 每根HiTrap Chelating 金属螯合柱可在少於25 分钟中纯化12 mg 融合蛋白包含所需浓缩缓冲液和工具使用针筒，蠕动泵，或者KTA 系统添加剂兼容HiTrap Chelating HiTrap Chelating Chelating Sepharose Fast Flow 适合自行装柱和放大检测( His)6 融合蛋白检测( His)6 融合蛋白其他融合或非融合蛋白的纯化(1/3) 如果有对应配基，使用亲和层析一步纯化高灵敏度高载量其他融合或非融合蛋白的纯化(2/3) 其他融合或非融合蛋白的纯化(3/3) 其他融合或非融合蛋白的纯化准备配基( e.g. 抗体) 准备柱子( e.g. NHS-activated HiTrap )  现成方法优化结合和洗脱条件使用针筒，蠕动泵，或者KTA 系统操作的HiTrap 小柱其他融合或非融合蛋白的检测去处小分子纯化后( His)6 融合蛋白的咪唑酶切后的GST 或His 标记多余的盐/尿素/盐酸胍用HiTrap Desalting 将样品转移到另一种缓冲液中，e.g. 储存用或改变pH 交换缓冲液和去盐的柱子快速有效高处理量去盐, 交换缓冲液, 去掉低分子量物质交换缓冲液和去盐的柱子选择纯化设备选择纯化设备多步纯化(非融合性蛋白或需要更高纯度) 工艺开发和优化法规需要e.g. GLP/GMP 纯化工艺放大转移工艺到更大规模大肠杆菌包涵体表达( His)6 融合蛋白的扩增，纯化和复性详细步骤过程包涵体- 细胞破碎, 冲洗和分离每100 ml 培养液重新悬浮细胞沉淀物於4 ml 20 mM Tris -HCl pH 8.0 在冰浴情况下，用超声震荡破碎细胞，并在+4o C 下高速离心10 分钟重新悬浮细胞沉淀物於2 ml 含e.g. urea 的冲洗缓冲液和再度超声震荡并在+4o C 下高速离心10 分钟用冲洗缓冲液冲洗细胞沉淀物溶解和准备样品重新悬浮细胞沉淀物於5 ml 溶解缓冲液( e.g.): 		20 mM Tris -HCl, 0.5 M NaCl,5 mM 咪唑, 6 M 盐酸胍,1 mM 2- 巯基乙醇pH 8 准备HiTrap Chelating 柱冲洗5 ml H2O 加0.5 ml 0.5 M NiSO4 冲洗5 ml H2O 用5-10 ml 20 mM Tris -HCl, 5 mM 咪唑, 6 M 盐酸胍,1 mM 2- 巯基乙醇pH 8 结合缓冲液洗柱纯化和复性组份分析多维蛋白质纯化高效纯化策略减少处理步骤准备工作考虑: 纯度水平需要量保持生物活性有效和经济蛋白质特性: 稳定性- pH - 离子强度- 温度分子量等电点pH 的重要性: 蛋白质净电荷随pH 改变pH 的重要性: 蛋白质净电荷随pH 改变样品准备考虑: 根据蛋白质来源选择不同萃取方法使用温和的步骤减少酸化和释放蛋白酶在室温以下快速处理用缓冲液维持pH, 离子强度目的: 稳定样品三步纯化策略三步纯化策略三步纯化策略三步纯化策略粗提目的纯化粗样快速浓缩(减少体积) 和稳定样品(去除蛋白酶) 最适用层析技术: 离子交换/ 疏水层析粗提: 离子交换填料粗提: 疏水层析填料高载量高流速HiPrep 16/10 Phenyl ( 高& 低取代) HiPrep 16/10 Butyl HiPrep 16/10 Octyl 中度纯化目的去除大部分杂质最适用层析技术: 离子交换/ 疏水层析中度纯化: 离子交换填料中度纯化:疏水层析填料精细纯化目的达到最终纯度(去除聚合物,结构变异物) 最适用层析技术: 凝焦过滤/离子交换/ 疏水层析/反相层析精细纯化: 凝胶过滤填料精细纯化: 凝胶过滤填料精细纯化:离子交换填料精细纯化:离子交换和疏水层析填料精细纯化:反相层析填料Source 适用於三步纯化策略的层析技术适用於三步纯化策略的层析技术适用於三步纯化策略的层析技术纯化工艺中不同层析技术的衔接一般准则: 结合互补选择性的技术，纯化工艺应尽量采用不同层析技术( e.g. IEX, HIC and GF) 减少不同层析技术间的样品处理( e.g. 浓缩, 交换缓冲液) 尽量简单衔接层析技术时注意事项合理衔接不同层析技术合理衔接不同层析技术合理衔接不同层析技术一个可溶性，非融合蛋白质的多维纯化DAOCS 去乙酰氧化头孢菌素C 合成酶DAOCS - 纯化策略DAOCS - 纯化目标得到5-10 mg DAOCS，纯度足够进行结晶和X-射线绕射分析在一个工作天内完成整个纯化流程工艺可放大最少10 倍. 结晶纯需要大分子均一性- 其他, 污染性蛋白质序列均一性- 蛋白水解片段- 转译后修饰	- 其他修饰(氧化等) 构象均一性- 聚合- 失活DAOCS - 定性DAOCS - 一般准则在所有缓冲液中加DTT 使所有半胱氨酸残基保持还原状态加入蛋白酶抑制剂以保持生物活性用SDS PAGE 检查每一个组份中DAOCS 利用酶测定法测量生物活性准备样品选择粗提的层析技术阴离子交换: 快速, 浓缩样品处理最简单分离基础: 电荷因为DACOS 的酸性pI 和不稳定性，所以缓冲液选择中性pH 粗提- 在KTA FPLC 上优化梯度选择中度纯化技术HIC: 分离基础: 疏水性蛋白质疏水性质很难预测: 筛选适合填料HIC 可以跟IEX 互补衔接, 减少样品处理步骤(只需要加盐) DAOCS 在高盐下能保持稳定选择精细纯化的层析技术GF: 分离基础: 分子量差异GF: 最适合分离双体，寡聚体和聚合物DAOCS - 优化好的粗提步骤DAOCS - 优化好的中度纯化步骤DAOCS - 优化好的精细纯化步骤DAOCS - 用SDS-PAGE 分析DAOCS 纯化- 结果DAOCS 纯化- 结果DAOCS 纯化- 总结KTA 平台层析系统纯化DAOCS - 结论DAOCS 纯度足够进行结晶使用KTA 平台层析系统的模板和快速探索(scouting) 功能可以快捷有效地优化纯化工艺纯化时间从3 天减少到6 小时请参考中文网站谢谢欢迎您随时和我们联系 support@apbiowww.book118.com 北京(010) 6487 3549上海(021) 5080 0260广州(020) 8732 0255香港(852) 28117575 Column and elpho conditions: Column:	GSTrap 1 ml Sample:	8 ml cytoplasmic extract from E. coli expressing a GST fusion protein Binding buffer:	PBS, pH 7.3 Elution buffer:	50 mM Tris -HCl, pH 8.0 with 10 mM reduced glutathione Flow rate:	1 ml/min Chromatographic procedure:	4 CV binding buffer, 8 ml sample, 10 CV binding buffer, 	5 CV elution buffer, 5 CV binding buffer (CV = column volume) System:	 KTA explorer 10 Lane 1:	Low Molecular Weight (LMW) Calibration kit, 	reduced, Amersham Pharmacia Biotech Lane 2:	Cytoplasmic extract of E. coli expressing 	GST fusion protein, 1 g cell paste/10 ml Lane 3:	GST fusion protein eluted from GSTrap 1 ml If a purification procedure is to be used several times, there is a lot to gain by strategic thinking and careful development work. Even with the reasonably yield of 80% in each purification step, one is down to approximately 20% overall yield after 8 purification steps. Limiting the number of steps in a purification procedure, and optimizing each step, thus pays off considerably. These three aims or requirements have to be met in essentially all protein purification situations. “Economy”in this context is related both to the cost of equipment and consumables, and to economic use of the start material (i.e. an acceptable yield) and to time and manpower needed. To obtain sufficient purity and quantity of the target protein, in an economic way, is a valid requirement both for production of a recombinant protein intended for therapy and production of an enriched protein extract for biochemical characterisation purposes. (But the meaning of “good economy”will be very different in these two extremes). The key to succesful protein purification is firstly to select the most appropriate techniques for the individual steps, and secondly to optimize the individual steps. This may sound simple, but what merits a separate lecture on the topic of protein purification strategies is the complexity of the input required to make the most clever selections. Titration curves will give the most complete picture of how the charge of the proteins in a sample vary with pH. In this example the titration curves of a sample containing three proteins is shown. Maximum separation can be expected at a pH where there is maximum separation between the titration curves for the individual proteins. Below the pI value of the red protein, all proteins are positively charged and will bind to cation exchangers, above the pI of the blue protein all proteins are negatively charged and will bind to an anion exchanger. The largest differences are found at about pH 3-4 and pH 9-10 and therefore cation or anion exchange chromatography might be a good choice at these respective pH values. But if we take into consideration the stability range of the proteins, in this example in the neutral to alkaline pH range, an anion exchange column at pH 9-10 will be the best choice. Chromatograms 1 and 2 show the protein sample run at different pH conditions on an anion exchanger. Chromatogram 1 was performed at a little lower pH and, as indicated by the titration curves, where the green and blue protein show similar charge at the pH chosen; no separation between these two is achieved chromatographically. At the second, more basic pH value, the titration curves are clearly separated and, as expected, a