Kapust R B, Waugh D S
Program in Structural Biology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, 21702-1201, USA.
Protein Expr Purif. 2000 Jul;19(2):312-8. doi: 10.1006/prep.2000.1251.
Here we describe a method for controlled intracellular processing (CIP) of fusion proteins by tobacco etch virus (TEV) protease. A fusion protein containing a TEV protease recognition site is expressed in Escherichia coli cells that also contain a TEV protease expression vector. The fusion protein vector is an IPTG-inducible ColE1-type plasmid, such as a T7 or tac promoter vector. In contrast, the TEV protease is produced by a compatible p15A-type vector that is induced by tetracyclines. Not only is the TEV protease regulated independently of the fusion protein, but its expression is highly repressed in the absence of inducer. Certain fusion partners have been shown to enhance the yield and solubility of their passenger proteins. When CIP is used as a purification step, it is possible to take advantage of these characteristics while both eliminating the need for large amounts of pure protease at a later stage and possibly simplifying the purification process. Additionally, we have observed that in some cases the timing of intracellular proteolysis can affect the solubility of the cleaved passenger protein, allowing it to be directed to either the soluble or the insoluble fraction of the crude cell lysate. This method also makes it possible to quickly gauge the efficiency of proteolysis in vivo, before protein purification has begun and in vitro processing is attempted.
在此，我们描述了一种利用烟草蚀纹病毒（TEV）蛋白酶对融合蛋白进行可控细胞内加工（CIP）的方法。含有TEV蛋白酶识别位点的融合蛋白在同时含有TEV蛋白酶表达载体的大肠杆菌细胞中表达。融合蛋白载体是一种IPTG诱导型ColE1型质粒，如T7或tac启动子载体。相比之下，TEV蛋白酶由一种兼容的p15A 型载体产生，该载体由四环素诱导。TEV蛋白酶不仅独立于融合蛋白进行调控，而且在没有诱导剂的情况下其表达受到高度抑制。某些融合伴侣已被证明可提高其客体蛋白的产量和溶解性。当CIP用作纯化步骤时，可以利用这些特性，同时消除后期对大量纯蛋白酶的需求，并可能简化纯化过程。此外，我们观察到，在某些情况下，细胞内蛋白水解的时机可影响裂解后客体蛋白的溶解性，使其定向进入粗细胞裂解物的可溶或不溶部分。该方法还使得在开始蛋白质纯化和尝试体外加工之前，能够快速评估体内蛋白水解的效率。
