Inhibition of tobacco etch virus protease activity by detergents
Inhibition of tobacco etch virus protease activity by detergents
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DOI：
10.1016/s1046-5928(02)00589-2
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发表时间：
2003-01-01
影响因子：
1.6
通讯作者：
Wiener, MC
中科院分区：
文献类型：
--
作者：
Mohanty, AK;Simmons, CR;Wiener, MC
多组氨酸等亲和标签极大地促进了重组蛋白的生产。整体膜蛋白的溶解度是通过蛋白质-洗涤剂复合物（PDCs）的形成来维持的，洗涤剂的浓度高于其临界胶束浓度（CMC）。去除亲和标签需要包含一个工程蛋白酶裂解位点。烟草蚀刻病毒（TEV）蛋白酶是一种常用的去除标签的蛋白酶。TEV以重组形式（rTEV）可用，并且通常包含其自身的多组氨酸亲和标签，用于酶消化后去除。标记结构域的蛋白水解裂解是通过将该蛋白与rTEV蛋白酶孵育进行的。我们观察到，在整体膜蛋白的纯化、结晶和其他生化研究中使用的各种洗涤剂存在时，rTEV消化的效率显著降低。蛋白酶活性的降低提示了洗涤剂对rTEV的抑制作用。为了验证这一假设，我们研究了洗涤剂对可溶性融合蛋白α(1)、血小板活化因子乙酰水解酶（PAFAHalpha(1)）的rTEV蛋白水解消化的影响。六组氨酸氨基末端亲和标签的去除在16种不同的去污剂浓度高于其各自的cmc的情况下进行了表征。我们的数据表明，测试的洗涤剂中有一半会降低rTEV的活性，这些洗涤剂应避免使用或在重组整体膜蛋白的rTEV消化过程中使用。(C) 2002 Elsevier Science （USA）。版权所有。
Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1), platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins. (C) 2002 Elsevier Science (USA). All rights reserved.