Larsen Anna K, De Veyra Teresa, Jia Zongchao, Wells Alan, Dutt Previn, Elce John S
Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, 3, DK-1958 Frederiksberg C, Denmark.
Protein Expr Purif. 2004 Feb;33(2):246-55. doi: 10.1016/j.pep.2003.10.005.
The two best known calpains, micro- and m-calpain, are Ca(2+)-dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca(2+)-linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (micro-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine-Dalgarno ribosome binding site. The results provide a simple method to obtain approximately 10mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.
两种最知名的钙蛋白酶，即μ-钙蛋白酶和m-钙蛋白酶，是存在于所有哺乳动物组织中的钙依赖性半胱氨酸蛋白酶。它们可能参与了许多与钙相关的信号通路，尽管具体细节尚不清楚。这些酶是由一个特定的大亚基（μ-80k或m-80k）和一个共同的小亚基（28k）组成的异二聚体。通过在大肠杆菌和Sf9细胞中共表达大亚基和小亚基，已获得重组钙蛋白酶，但成功率各不相同。与28k亚基一起表达时水平很低，但与该亚基的C末端21k片段一起表达时则高得多。大鼠m-钙蛋白酶（m-80k/21k）在大肠杆菌中表达良好，但小鼠m-钙蛋白酶（m-80k/21k）表达不佳，尽管大鼠m-80k和小鼠m-80k的氨基酸序列有92%的同一性。也有报道称人m-钙蛋白酶可在Sf9细胞中表达，但不能在大肠杆菌中表达。为了研究这些差异，构建了大鼠/小鼠和大鼠/人杂交的m-钙蛋白酶并在大肠杆菌中表达。结果表明，小鼠m-80k序列特有的异亮氨酸-6和脯氨酸-127导致表达不佳。通过提供正确的SD核糖体结合位点，可以在大肠杆菌中实现人m-钙蛋白酶的高表达。这些结果提供了一种简单的方法来获得约10mg的人m-钙蛋白酶和一种略有修饰的小鼠m-钙蛋白酶。还研究了m-80k-EGFP融合蛋白在大肠杆菌和哺乳动物细胞中的表达情况，同时改变小亚基和启动子。单独的m-80k-EGFP没有活性，但与21k或28k亚基一起时在两种细胞类型中都有活性。EGFP结构域在表达过程中被部分切割，释放出有活性的m-80k/21k钙蛋白酶。
