蛋白质与蛋白组学

1.10. Solubility on overexpression in E. coli
Several studies have proposed that one can predict from the protein sequence the likelihood that a protein, if overproduced in E. coli will be soluble or form insoluble inclusion bodies (Idicula-Thomas and Balaji, 2005; Wilkinson and Harrison, 1991). Obviously, this is useful information, but is most easily obtained by simply overproducing a protein, breaking the cells, centrifuging the lysate to separate soluble from insoluble, and then analyzing the two resulting fractions by SDS–PAGE. The fact that the proportion of an overproduced protein that is soluble can be significantly altered by manipulating cell growth conditions suggests that solubility predictions based on sequence may be useful but have severe limitations.

大肠杆菌过表达的蛋白是可溶还是形成包涵体？有一些研究提出可以通过蛋白质的序列来预测其可能性。显然，这种信息很有用。但是，过表达一下蛋白、破菌、离心，然后分别对上清和沉淀做SDS-PAGE分析，这样也许更为简单实用。原因在于过表达蛋白中可溶性的比例受到细菌培养条件的极大影响。这也说明虽然通过序列来预测可溶性有些用处，但有严重的缺陷性。

不错，不错，又一个用实践战胜理论的例子。培养条件对可溶性的影响做过原核表达的应该都是深有体会。我们经常为了得到可溶性的蛋白不断对培养和诱导条件做优化，例如温度、摇床转速、诱导时菌体密度、诱导剂浓度、诱导时间等等。

文章没有指出的是，实际上不仅是培养条件，不同的宿主菌，不同的质粒等都有影响。还有一个非常重要的因素常常被大家所忽略，那就是破菌缓冲液和破菌条件。我们是通过电泳来检测是否可溶的，而电泳样品来源于破菌上清和破菌沉淀，这就与破菌缓冲和破菌方式肯定相关了。在一种破菌缓冲液，如PBS中，破菌电泳检测为不可溶的蛋白，在另一种破菌缓冲液，如pH8.0的Tris中，破菌检测很可能就是可溶的了。

2. What You Cannot yet Predict
那些我们无法预测
2.1. Three-dimension structure; shape, surface features
While a huge amount of time and effort has gone into finding a practical method of predicting three-dimensional structure from amino acid sequence, it is presently not yet possible to do so with any confidence. In the case where a target protein shows high similarity to a protein whose structure has been determined, it is possible to ‘‘thread’’ the sequence into the known structure and come up with a reasonable approximation of the structure of the target protein.

三维结构、形状和表面特征
虽然人们花费了大量的时间和努力，想要找到由氨基酸序列预测三维结构的实用方法，目前来说仍然没有多大可信度。如果案例中的目标蛋白表现出与已知结构蛋白有很高的类似性，那么将目标蛋白的序列“装入”到已知结构中，从而得到目标蛋白结构的合理近似。

Without accurate structural information, one cannot predict shape, or detailed surface features such as hydrophobic patches, charge distribution, and antigenic sites. Therefore, one cannot easily predict behavior on hydrophobic interaction chromatography (HIC) or ion exchange chromatography. Since the shape affects the Stokes radius, one cannot predict behavior during gel filtration chromatography (a spherical protein will appear smaller than an asymmetric or cigar-shaped protein of the same MW during gel filtration).

没有精确的结构信息，就不能预测形状或详细的表面特征，如疏水区域，电荷分布，抗原表位等。进一步说，就不能方便地预测其疏水层析（HIC）或离子交换层析的柱行为。因为形状影响到分子的Stoke半径，所以也不能预测凝胶过滤层析的柱行为（在凝胶过滤中，同样分子量大小的蛋白，球形蛋白比非对称蛋白或雪茄形蛋白表现的要小一些）。

理论讲得不错。就凝胶过滤来说，文章的这个理由——形状的不同，可能对实际纯化操作意义不大。一般凝胶过滤的分辨率都不是很高，两个蛋白要分开，其MW的差异可能要一倍以上。所以，二硫键、多聚体等因素的影响更为重要。

2.2. Multisubunit features; homomultimers, heteromultimers?
Even if one could accurately predict the three-dimensional structure of a protein, it would still be impossible to predict if it exists in solution as a multimer (e.g., a hexamer) or as a monomer. That lack of knowledge precludes any reasonable prediction of its behavior on sizing columns or on ion exchange columns. Even more problematic is the fact that many
proteins exist as parts of multisubunit complexes and one cannot predict whether a given protein will exist as a complex whose purification properties may be determined largely by its binding partners.

多亚基特征，均相多聚体，非均相多聚体？
即使人们能够准确预测一个蛋白的三维结构，也仍然不可能预测到它在溶液里是以多聚体（如六聚体）存在，还是以单体形式存在。此类知识的缺乏阻碍了对目标蛋白在分子筛柱或离子交换柱上的行为做任何合理的预测。事实上，更为严重的问题是，很多蛋白是作为多亚基复合物的部分存在的，但人们不能预测出一个给定蛋白是否作为复合物存在的，而很可能复合物的纯化性质主要由它的结合伙伴们所决定。

后一句比较绕口，简化一下：
1、蛋白A，蛋白B，C等可能形成复合物，我们不知道真实情况；
2、目标纯化蛋白A，通过某种纯化方法（例如针对A的亲和层析）无法纯化，很可能就得到没有A的错误结论；
3、目标纯化蛋白A，也许可以通过针对蛋白B或C的亲和层析方法得到A。

2.3. Precipitation properties
Different proteins vary in their solubility properties in ways that are not yet understood. Therefore, it is not presently possible to predict what ammonium sulfate concentration to use in precipitating a given protein from solution. This will be discussed further in Chapter 20.

沉淀性质
不同的蛋白溶解性不同，但他们是如何变化的还是不了解。因此，目前还不能够预测沉淀给定溶液中蛋白的硫酸铵浓度。在第20章中将做进一步的讨论。

曾经看到过一个求助，问针对某个分子量蛋白的硫酸铵沉淀的浓度是多少，这是一个说明了。没有一个所谓的分子量与硫酸铵浓度的关系存在，做梯度试验是唯一途径。

3. Conclusion
I think it is very clear that one cannot yet use amino acid sequence to predict the behavior of a given protein in most of the primary fractionation methods used by protein purifiers. By far the best approach still is to fractionate an extract by ammonium sulfate precipitation, by ion exchange and gel filtration chromatography and determine how the protein of interest behaves. Therefore, although we have tremendous knowledge of protein sequence, protein purification is still very empirical! Don’t be afraid to simply do the experiment!

结论
情况已经很明了，在绝大多数蛋白质纯化人员所使用的基本纯化方法中，通过氨基酸序列来预测给定蛋白的纯化行为仍然是行不通的。到目前为止，最佳的方案依然是通过硫酸铵沉淀、离子交换和凝胶过滤层析来分离提取物，确定目标蛋白是如何表现的。因此，虽然我们有着海量的蛋白质序列知识，但蛋白质纯化依然是一项非常经验性的工作！不要怕，去做好了。

3.1. Protein bioinformatic resources
蛋白质生物信息学工具

One of the most used sites for obtaining sequence data and using it to compute various physicochemical properties of proteins is the ProtParam feature of ExPASy (Expert Protein Analysis Software)Web site (cuturl('http://www.expasy.org/tools/protparam')). ProtParam calculates many of the protein parameters including MW, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (Gasteiger et al., 2005).

使用最多的网站之一是ExPASy(Expert Protein Analysis Software)网站中的ProtParam部分 (cuturl('http://www.expasy.org/tools/protparam'))，用它可以得到蛋白质的序列数据，还可以计算蛋白质各种各样的理化性质。ProtParam能够计算的蛋白质参数有：分子量MW，等电点pI，氨基酸组成，原子组成，消光系数，估算半衰期，不稳定性系数，脂溶指数和总平均亲水性。

对脂溶指数（aliphatic index）和总平均亲水性（Grand Average of Hydropathy，GRAVY）的概念不清楚，专门到Expasy站点去看了看，定义如下：cuturl('http://www.expasy.ch/tools/protparam-doc.html')
The aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine). It may be regarded as a positive factor for the increase of thermostability of globular proteins. The aliphatic index of a protein is calculated according to the following formula:
Aliphatic index = X(Ala) + a * X(Val) + b * ( X(Ile) + X(Leu) )
where X(Ala), X(Val), X(Ile), and X(Leu) are mole percent (100 X mole fraction) of alanine, valine, isoleucine, and leucine.
The coefficients a and b are the relative volume of valine side chain (a = 2.9) and of Leu/Ile side chains (b = 3.9) to the side chain of alanine.

简单的讲脂溶指数就是蛋白质脂肪侧链占蛋白质的相对含量，由蛋白质中Ala，Val，Ile，Leu的含量所决定。被认为代表了蛋白质的热稳定性，但是多大的数值表示稳定呢？我还不知道。从一篇文章中看到(aliphatic index)为75.50, 表明其为脂溶蛋白。
GRAVY (Grand Average of Hydropathy)
The GRAVY value for a peptide or protein is calculated as the sum of hydropathy values of all the amino acids, divided by the number of residues in the sequence.
定义为序列中所有氨基酸亲水值的总和与氨基酸数量的比值，负值越大表示亲水性越好好，正值越大表示疏水性越强。

This is very simple to use. Go to ProtParam tools, enter a Swiss-Prot/TrEMBL accession number (AC) or a sequence identifier (ID), or you can paste your own sequence in the box, hit button that says ‘‘compute parameters,’’ and print out the results.

使用很简单。进入ProtParam工具，输入Swiss-Prot/TrEMBL登录号，或序列识别号，或将序列复制到指定框中，单击“参数计算”，输出结果。

One commercial package available through DNASTAR (cuturl('http://www.dnastar.com')), called Lasergene, version 8.0, contains a section on protein sequence analysis called ‘‘Protean.’’ This allows sequence analysis similar to ProtParam and in addition, predicts antigenicity, surface probability, maps proteolytic digestion sites, and has very nice graphical displays.

一个商业化软件，叫做Lasergene，可以从DNASTAR (cuturl('http://www.dnastar.com'))网站得到，它含有一个叫“Protean”的蛋白质序列分析部分。它的序列分析与ProtParam相似，另外，它还可以预测抗原性、表面可能性、蛋白酶解位点图，还有很友好的图形展示。

为了增加感性认识，找了几篇中文的序列分析方面的文章：
运用生物信息学的方法对胶质纤维酸性蛋白的初步分析.pdf
HCV p7蛋白反式调节基因p7TP2的克隆化及生物信息学分析.pdf
一个白菜花粉发育相关的新基因BcMF7 的鉴定.pdf
放射性碘标记人工合成RGD 肽拮抗物的设计与分子特性的计算机分析与模拟研究.pdf

感兴趣的可以用Google搜一下，读一读，理解会更深一些。

3.2. Purification in the denatured state
After all that was discussed above, it is only fair to point out that one can do much better at predicting purification properties of a given protein is you are willing to carry out the purification in the denatured state (Knuth and Burgess, 1987). If you do not have to worry about possible multimeric states, then one can simply denature in urea, fractionate somewhat predictably by ion exchange and gel filtration, and then refold the protein to its native structure (see Chapter 17).

变性状态下纯化
略。

对此段有保留意见。采用尿素变性纯化，不一定会使得柱行为更能预测，不管是离子交换还是凝胶过滤。变性后再进行复性也不是一个容易的事。simply denature带来的后果可不是simple。

2. Amount of total protein (mg).
This is usually determined by a standard protein assay. Most commonly these days a Bradford dye-binding assay or a bicinchoninic acid (BCA) assay is used (see Chapter 8). It is important to indicate in the methods section what protein is used as the protein standard (typically BSA). Once the protein is purified it can also sometimes be quantified by measuring its absorbance at 280 nm and the use of an appropriate molar extinction coefficient.

总蛋白量（mg）
一般用标准蛋白分析方法来确定总蛋白量。现在最常用的方法是Bradford和BCA。在方法章节讨论了用何种蛋白作为标准蛋白（常用BSA）的重要性。一旦蛋白纯化好了，有时候测定280nm的紫外吸收值，再应用其摩尔消光系数就可以进行蛋白定量。

蛋白质定量从本质上讲是一个确定的值，质量数吗？好像应该是一定。但是由于定量方法的缺陷，无论是那种方法都有很多干扰，所以只能是一个相对的值。就像文中所讲，标准蛋白很重要，最好使用与待测蛋白相同的蛋白做标准，但这又不现实，所以通常都用便宜、易得，且比较稳定的BSA来做标准蛋白了。

在工作中，要注意的是保持方法的稳定性，不求最真，但求偏差恒定。换句话说，即使我测定的蛋白浓度比真值偏差50%，以后一直保持这个50%的偏差对实际工作来说多数时候是可以接受的。