Two-promoter vector is highly efficient for overproduction of protein complexes
Two-promoter vector is highly efficient for overproduction of protein complexes
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DOI：
10.1110/ps.04644504
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发表时间：
2004-06-01
期刊：
影响因子：
8
通讯作者：
Oh, BH
中科院分区：
文献类型：
--
作者：
Kim, KJ;Kim, HE;Oh, BH
使用双顺反子载体，其在一个启动子下含有两个靶基因，是异源生产二元蛋白质复合物的最常见实践。该方法的主要问题是与启动子旁边的第一基因相比，第二基因的表达低得多。我们测试了一个简单的想法，即在第二个基因前面包含一个额外的启动子是否可以解决这个问题。与双顺反子载体相比，相应的双启动子载体在细菌细胞中产生4至9倍更大量的BCL-2家族蛋白BCL-X-L：BAD、BCL-X-L、XL：BIM-S和CED-9：EGL-1之间的复合物，这是第二基因以与靶基因的顺序无关的方式显著增加表达的结果。用双促进剂体系，我们大量生产了另外两种适合于广泛结晶试验的配合物。该方法不伴随任何技术缺点，并且代表了从常规方法的显著改进，该常规方法应该通过扩展而在二元或更高阶蛋白质复合物的共表达中享有广泛的应用。
The use of bicistronic vectors, which contain two target genes under one promoter, has been the most common practice for the heterologous production of binary protein complexes. The major problem of this method is the much lower expression of the second gene compared with that Of the first gene next to the promoter. We tested a simple idea of whether inclusion of an additional promoter in front of the second gene may remove the problem. Compared with bicistronic vectors, corresponding two-promoter vectors yielded four to nine times larger amounts of the complexes between BCL-2 family proteins, BCL-X-L:BAD, BCL-X-L, XL:BIM-S, and CED-9:EGL-1 in bacterial cells as a result of significantly increased expression of the second genes in a manner independent of the order of the target genes. With the two-promoter system, we produced two other complexes in large quantity suitable for extensive crystallization trial. The method does not accompany any technical disadvantages, and represents a significant improvement from the conventional method, which should enjoy wide application for the coexpression of binary or higher order protein complexes by extension.