Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro
Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro
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DOI：
10.1016/j.pep.2004.08.016
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发表时间：
2004-11-01
影响因子：
1.6
通讯作者：
Waugh, DS
中科院分区：
文献类型：
--
作者：
Nallamsetty, S;Kapust, RB;Waugh, DS
亲和标签被广泛用作生产重组蛋白的载体。然而，由于担心它们可能干扰蛋白质的活性或结构，将它们从目标蛋白质中移除几乎总是可取的。最常用于切割融合蛋白的酶是凝血因子Xa、肠激酶和凝血酶，然而文献中充满了关于这些酶在设计位置以外的位置切割融合蛋白的报道。越来越明显的是，某些病毒蛋白水解酶具有更严格的序列特异性。这些酶采用类胰酶折叠，但具有非传统催化三联体，其中半胱氨酸取代丝氨酸。烟草蚀刻病毒(TEV)蛋白酶是这类酶中特性最好的一种。TeV蛋白酶将序列ENLYFQG/S切割在QG或QS之间，具有很高的特异性。烟草静脉斑驳病毒蛋白水解酶是烟草静脉斑驳病毒蛋白水解酶的近亲，具有明显的序列特异性(ETVRFQG/S)。我们发现，与TEV蛋白酶一样，TVMV蛋白酶在体外和体内都可以用来切割具有高度特异性的融合蛋白。我们以MBP-NusG融合蛋白为模型底物，比较了两种酶的催化活性随温度和离子强度的变化。TVMV蛋白水解酶的行为与TEV蛋白水解酶非常相似。在没有氯化钠的情况下，其催化活性最高，但当盐增加到200 mM时，其催化活性仅下降三倍。我们发现，这两种酶的最适温度几乎相同，在室温和4℃下的催化效率只有两倍之差。因此，当重组蛋白碰巧含有与TEV蛋白酶识别位点相似的序列时，TVMV蛋白酶可能是TEV蛋白酶的有用替代品，或者对于涉及使用多种蛋白酶的蛋白质表达策略来说，TVMV蛋白酶可能是有用的替代。由爱思唯尔公司出版。
Affinity tags are widely used as vehicles for the production of recombinant proteins. Yet, because of concerns about their potential to interfere with the activity or structure of proteins, it is almost always desirable to remove them from the target protein. The proteases that are most often used to cleave fusion proteins are factor Xa, enterokinase, and thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the designed site. It is becoming increasingly evident that certain viral proteases have more stringent sequence specificity. These proteases adopt a trypsin-like fold but possess an unconventional catalytic triad in which Cys replaces Ser. The tobacco etch virus (TEV) protease is the best-characterized enzyme of this type. TEV protease cleaves the sequence ENLYFQG/S between QG or QS with high specificity. The tobacco vein mottling virus (TVMV) protease is a close relative of TEV protease with a distinct sequence specificity (ETVRFQG/S). We show that, like TEV protease, TVMV protease can be used to cleave fusion proteins with high specificity in vitro and in vivo. We compared the catalytic activity of the two enzymes as a function of temperature and ionic strength, using an MBP-NusG fusion protein as a model substrate. The behavior of TVMV protease was very similar to that of TEV protease. Its catalytic activity was greatest in the absence of NaCl, but diminished only threefold with increasing salt up to 200 mM. We found that the optimum temperatures of the two enzymes are nearly the same and that they differ only two-fold in catalytic efficiency, both at room temperature and 4degreesC. Hence, TVMV protease may be a useful alternative to TEV protease when a recombinant protein happens to contain a sequence that is similar to a TEV protease recognition site or for protein expression strategies that involve the use of more than one protease. Published by Elsevier Inc.