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蛋白表达纯化之通关技巧

发布: 2019-07-29 993阅读 173评论

拿到一个蛋白以后，首先需要对蛋白进行全面的了解，所谓知彼知己方能百战不殆：

1. 蛋白二级结构预测：蛋白质的二级结构可以提供蛋白质的 α 螺旋、β 折叠和柔性区的分布情况。
http://www.compbio.dundee.ac.uk/jpred/

2. 跨膜区预测：蛋白质的跨膜区的位置预测有助于我们了解蛋白质共有几次跨膜，确定胞外域和胞内域的位置。
http://www.cbs.dtu.dk/services/TMHMM-2.0/

3. 信号肽预测：蛋白质的信号肽预测有助于我们了解蛋白质的一些特性，例如蛋白是否为分泌蛋白。
http://www.cbs.dtu.dk/services/SignalP/

4. 糖基化位点预测：糖基化位点预测，在蛋白的表达和结晶过程中有重要参考意义。
http://www.cbs.dtu.dk/services/NetOGlyc/

5. 理化性质分析：蛋白质的理化性质有助于我们了解蛋白质的部分特性，有助于蛋白质的纯化。
https://web.expasy.org/protparam/

6. 亲水性疏水性分析：分析蛋白的亲水性和疏水性，为选择纯化方法提供参考。
https://web.expasy.org/cgi-bin/protscale/protscale.pl

俗话说的好，能表达的蛋白千篇一律，不表达的蛋白各有不同。这蛋白不表达的原因有很多，可以从以下几点考虑：

载体构建是否合适——检查启动子、读码框、终止子等。某些基因含有许多稀有密码子，尤其是起始密码子后15 个碱基内的，会显著影响蛋白的表达。

表达系统是否恰当——常见的有四大表达系统，即大肠杆菌、酵母细胞、昆虫细胞以及哺乳细胞表达系统。不同的表达系统适合表达不同的蛋白，这个要根据情况选择。

标签选择是否合适——GST、SUMO 等标签具有提高融合蛋白可溶性的能力。

实验步骤是否正确——细节决定成败，一个操作上的小失误，可能直接影响最终结果。

其次，重组蛋白表达系统的选择也是关键的一步！

表1. 重组蛋白表达系统推荐

最后一步就是将表达的蛋白进行纯化。选择合适的纯化方法，可以提高蛋白的纯化效率，让蛋白纯化简单便捷。常用的方法有:亲和层析、离子交换层析、凝胶过滤层析、疏水层析等。

亲和层析
亲和层析纯化是利用生物大分子物质具有与某些相应的分子专一性可逆结合的特性进行蛋白纯化的技术。

离子交换层析
离子交换层析是一种依据蛋白表面所带电荷量不同进行蛋白分离纯化的技术。

凝胶过滤层析
凝胶过滤(也叫排阻层析或分子筛)是一种根据分子大小从混合物中分离蛋白质的方法。

疏水作用层析
疏水作用层析是利用盐-水体系中样品分子的疏水基团和层析介质的疏水配基之间疏水力的不同而进行分离的一种 层析方法。

表2. 不同层析方法对比

通过上述方法，不知道大家有没有掌握蛋白表达和纯化的方法~

详情请咨询当地销售或致电15320177155，也可发送您的意向到 lisida@novogene.com。

参考文献
[1] Banos G, Coffey M P, Wall E, et al. Protein expression and purification[J]. Protein Expression & Purification, 1990, 57(2):206–216.
[2] Ghaemmaghami S, Huh W K, Bower K, et al. Global analysis of protein expression in yeast.[J]. Nature, 2003, 425(6959):737-741.

结构生物学事业部 霍洁蕊、王 宇 | 文案
孙津津丨编辑
图片来源于网络，侵删

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