蛋白质与蛋白组学

策略Strategies，又见策略。呵呵，其实所谓策略是最简单的、共性的东西；是方向性的，但不是具体性的；可以用来指导试验的设计，但不能指导试验的细节；是基础知识，不是高楼。
我们要从策略中学习到轮廓性的东西，在自己的试验当中去清晰它。

It is well worth the effort to carry out stability studies (e.g., heat inactivation or storage trials) in order to learn how to maintain a stable, soluble protein. Three notes of caution: (1) optimal storage conditions may change with purification; (2) storage conditions may alter peptide–peptide interactions; and (3) optimal storage conditions need not relate to optimal conditions for activity. Indeed, materials that stabilize a protein might inhibit it when added to activity mixes. Of course, the latter situation must be considered when utilizing the protein—interfering substances will have to be removed or ‘‘diluted out’’ for utilization of the protein.

很有启发性的一段话。
1、蛋白质的最优储存条件可能会随着纯化过程的进展而变化；
2、蛋白质的储存条件可能会改变蛋白质之间的相互作用；
3、蛋白质的最优储存条件并非最佳的活性表现条件。

In our experience, reducing agents are particularly effective for storage of bacterial enzymes that often derive from a reducing environment, whereas enzymes from animals often take kindly to surfactants or protease inhibitors. Fungal proteins also respond to protease inhibitors. Optimal pH and salt concentrations vary widely.

细菌的酶产生于还原环境（文章的后面有叙述），所以含还原剂的保存条件对这种菌源酶特别有效。类似的，动物源的酶通常耐受表面活性剂或蛋白酶抑制剂；真菌源的酶对蛋白酶抑制剂也耐受。而保存最佳的pH和盐浓度却是个性相关，变化很大。

这段话也比较有意思，我以前从来没有意识到。现在看到了，但不是很认同。细菌的酶产生于还原环境，所以保存时应该加入还原剂？且不说细菌的酶是否都处于还原环境中。一个蛋白高级结构的维持是表现活性的必要条件。还原或非还原环境影响的是半胱氨酸之间形成二硫键。如果结构中的活性不依赖于自由的巯基（当然很多酶是依赖的，但也有很多是不依赖的），这就与还原无关，加入还原剂甚至有害。

这个表格不错，脑子中虽然有这些概念，但不如这个表格来的清晰。平常做纯化大约也就用到这些东西吧。不爽的是表格偷懒用Stability代替了不同的影响，比如说沉淀、疏水聚集、末端水解、酶水解等等。

Concerning the containers utilized for purified proteins, particularly at low concentrations, plastic rather than glass should be used. In our experience, polypropylene is superior to polyethylene or polystyrene and other clear plastics. In rare instances, we have had to precoat storage tubes with an inert protein prior to their use. Be sure to have tight-fitting caps if storage is in ‘‘frost-free’’ freezers. These and other precautions are discussed in detail in Chapter 10.

装纯化好的蛋白，特别是低浓度的蛋白，用塑料容器比用玻璃容器好。聚丙烯塑料容器好于聚乙烯、聚苯乙烯和其他透明塑料容器。在某些极端情况下，存储管在使用前要预包被一种惰性蛋白。

If tags are to be attached to a protein to aid in purification and/or detection, will they interfere with subsequent studies? Where in the protein should the tags be placed? If on the N-terminus, prematurely terminated peptides will contaminate affinity-purified material. If on the C-terminus, functional or binding properties of that part of the protein might be compromised (see Chapter 16 for discussion of this important topic).

如果目标蛋白加了纯化或检测用的tag，他们是否会影响后续的研究呢？tag应该放在目标蛋白的什么位置？放在N端，表达提前终止的片段会干扰亲和纯化产物。放在C端，目标蛋白的功能或结合活性可能会减弱。

有意思的一个论述。对重组蛋白的构建有一定的启发意义。这里面隐含了一个很重要的现象，一般蛋白质的C端有限的几个氨基酸是影响蛋白活性的重要因素。

4. Bulk or Batch Procedures for Purification
These procedures are almost always utilized early in the purification as they are usually most effective in removing non protein material and are amenable to the relatively large volume and amounts of material that exist in earlier stages of the preparation. A great deal of effort went into designing these steps in the early days of protein chemistry, and much frustration, time, and money can often be avoided by including some of these ‘‘old fashioned’’ procedures.

大量或批量蛋白纯化的步骤
纯化早期，常用这些手段来有效去除非蛋白类物质。在制备初期，大体积样品，高非蛋白物质含量用这些手段常常很实用。在蛋白质化学的早期阶段，前人做了大量的工作来完善这些批量纯化手段，那些费力、费时间和费钱的纯化过程如果包括这些“老式”的纯化手段常常可以得到改善。

Section V details many of these procedures. Gentle procedures including ‘‘salting out’’ or phase partition with organic polymers are most common. More drastic methods such as heat, extremes of pH, or phase partition with organic solvents might be particularly effective with stable proteins, though subtle forms of damage may be difficult to foresee or to detect. In addition, one might consider the use of high-capacity ion-exchange resins either added as a slurry to crude material or used in columns for batch elution. The time expended in developing and optimizing these early steps is always worthwhile—even removal of half of the contaminating protein may dictate the feasibility of a subsequent step from both cost and technical considerations.

第五节详述了这些“老式”的方法。温和的方法常用的有用有机高分子聚合物的“盐出”或相分配。更为剧烈的方法，例如加热、极端pH，或用有机溶剂的相分配，对某些稳定性的蛋白可能特别有效，尽管细微的损害难以预测或检测到。另外，可以考虑应用高容量的离子交换介质，可以直接作为胶浆加入到粗原料中，或者装柱做批次纯化洗脱。花时间研究优化这些早期步骤往往很有回报，即使只去除了一半的杂质蛋白，对后续步骤的成本和技术考虑都是很有益。

对头。对纯化来说，技术新，技术高并不代表是实用的好技术。这一点往往被我们忽视了。做蛋白质纯化，现在的试验室动则HPLC，制备电泳，密度离心，SOURCE，RESOURCE，用高分辨率的仪器，用高分辨率的纯化介质等等。不选对的，只选贵的。一个纯化工艺中如果有着很普通的胶，很简单的加热、沉淀等手段好像就不能显示纯化水平了，文章就低档次了，唉。
如果能根据目标蛋白的性质，选取用到有机溶剂萃取、沉淀（pH、硫酸铵、PEG等）、热处理等简单的方法对纯化大大有益。因为这些都是能够很方便放大的工艺方法。

Two notes of caution: it has been our experience that rapid dilution of a fraction that has a high concentration of salt, glycerol, or sucrose often results in a loss of activity. If this phenomenon is experienced, the use of dialysis or gel exclusion chromatography may be a useful alternative. Also, during a purification scheme, the protein solution sometimes becomes cloudy. This solution must be clarified prior to chromatography to avoid unacceptably low flow rates. In our experience, the insoluble material rarely contains native protein.

两点注意：其一，含高浓度盐、甘油或蔗糖的蛋白样品，快速稀释常常会导致活性损失。如果你的样品有此现象，改用透析或凝胶分子筛层析吧。其二，在纯化过程中，蛋白质溶液有时候会变浑浊，这种浑浊溶液在上柱层析之前应当澄清，以避免不得不用的低流速。以经验而言，不溶性的物质很少含有天然活性蛋白。

其一点我还真没有想到过，学习了。常用快速稀释法来进行包涵体的复性操作，没想到快速稀释对有活性的蛋白有时候是不利的。
其二点毫无讨价还价的余地。层析之前样品是一定要过滤的，这不仅是低流速的问题，还涉及到层析设备和层析柱的使用规范。我们一般都是离心加过滤。

It would seem that knowing the amino acid sequences would make it much easier to design an effective purification of that protein. We examine in this chapter what you can and cannot predict from an amino acid sequence and conclude that protein purification is still largely an empirical science.

知道目标蛋白的氨基酸序列对设计一个成功的纯化程序很有利。本章探讨通过氨基酸序列我们能够预测什么。最后要说的是，蛋白质纯化在很大程度上依然还是一门经验科学。

与我心有切切焉！empirical science是更好听一些了，加了一个science，呵呵。原来常说的是art，更传神一点。所谓art，那么就没有一个肯定的判断标准，在不同的人眼里有着不同的art。一幅画，一段音乐，可能是视觉污染，噪音，也可能是力作，是天籁。做蛋白质纯化，纯化经历不同的人，所开发出来的纯化工艺也各不相同。共性点有，但个性点更多。

1.1. Molecular weight of the polypeptide chain
It is very straightforward to calculate the molecular weight (MW) of a polypeptide chain protein by multiplying the MW of an amino acid in a polypeptide (the MW of an amino acid –18 Da) by the number of times that amino acid occurs in the polypeptide. Remember to add an extra 18 Da to the total to account for the free amino and carboxy termini. Of course, one cannot know, except by N-terminal sequencing or mass spectroscopy, whether the N-terminal initiating methionine has been removed or not.

多肽链的分子量
（略）

18 Da 是肽键形成时的水分子量。N端的甲硫氨酸问题对纯化无大碍。通过这么加加减减得到的是蛋白质的理论分子量。这个理论分子量的大小与电泳中的表观分子量的大小常常有差异，有时候还很大，但理论分子量的大小与质谱结果常常吻合的很好。另外，这个理论分子量没有考虑序列中特定位点的修饰问题，例如磷酸化、糖基化等。