小中大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。