Strategies to enhance the coexpression of cytochrome P450 2E1 and reductase in bacteria.
Strategies to enhance the coexpression of cytochrome P450 2E1 and reductase in bacteria.
复制标题
增强细菌中细胞色素 P450 2E1 和还原酶共表达的策略。
DOI：
10.1081/dmr-100101912
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Chang,S
中科院分区：
文献类型：
--
作者：
Porter,TD;Chang,S
哺乳动物的P450细胞色素包括一个超家族的膜结合血红素蛋白参与氧化的各种外源性和内源性物质。虽然哺乳动物P450在大肠杆菌中的表达已被证明是表征这些酶的有价值的手段，但相对于其他哺乳动物P450同种型，人细胞色素P450 2 E1在该生物体中表达较差，因此，几个研究组已探索了增加其表达的机制[1，2]。提高E.大肠杆菌已经对蛋白质的N-末端区域进行了修饰，通常删除[2-5]或替换[5-9]前8-21个残基。尽管没有证据表明这些修饰改变了表达蛋白的催化活性或特异性，但仍然存在担忧，一些研究人员已经采用了不需要修饰N-末端以实现有效表达的表达系统，如杆状病毒[10-13]。为了解决这些问题，我们尝试增加人细胞色素P450 2 E1的细菌表达，而不修饰N-末端，使用表达载体pINIIIompA 3 [14]，其融合了来自E. coli外膜蛋白A（ompA）连接到克隆蛋白的N末端。这种21个氨基酸的信号有效地将新生蛋白质引导到细菌的内膜，然后通过位于膜周质表面上的信号肽酶将肽去除。虽然最初开发的目的是促进异源蛋白分泌到周质中，但膜结合蛋白锚定到内膜上而不分泌。该载体已成功用于表达NADPH-细胞色素P450还原酶[15，16]，并且可能对具有弱膜插入信号序列的其他蛋白质有价值。在这里，我们表明，这种信号肽的人P450 2 E1，从pINIIIompA 3的高拷贝衍生物表达的N-末端的添加，产生了7倍的增加，在2 E1全酶从载体缺乏这一功能。
Mammalian P450 cytochromes comprise a superfamily of membrane-bound hemeproteins involved in the oxidation of a wide variety of exogenous and endogenous substances. Although expression of mammalian P450s in Escherichia coli has proven to be a valuable means to characterize these enzymes, human cytochrome P450 2E1 is poorly expressed in this organism relative to other mammalian P450 isoforms, and, thus, mechanisms to increase its expression have been explored by several groups [1, 2]. The most common approach to increasing P450 expression in E. coli has been to modify the N-terminal region of the protein, often deleting [2–5] or replacing [5–9] the first 8–21 residues. Although there is no evidence that these modifications alter the catalytic activities or specificities of the expressed proteins, concern remains nonetheless, and some investigators have adopted expression systems such as baculovirus that do not require modification of the N-terminus for efficient expression [10–13]. To address these concerns, we have attempted to increase bacterial expression of human cytochrome P450 2E1 without modifying the N-terminus, using an expression vector, pINIIIompA3 [14], that fuses the cleavable signal peptide from the E. coli outer membrane protein A (ompA) to the N-terminus of the cloned protein. This 21-amino acid signal efficiently directs nascent proteins to the inner membrane of the bacterium, where the peptide is then removed by the signal peptidase located on the periplasmic surface of the membrane. Although originally developed to promote the secretion of heterologous proteins into the periplasm, membrane-bound proteins become anchored to the inner membrane and are not secreted. This vector has been successfully used to express NADPH–cytochrome P450 reductase [15, 16] and may be valuable for other proteins with weak membrane insertion signal sequences. Here, we show that the addition of this signal peptide to the N-terminus of human P450 2E1, expressed from a high copy derivative of pINIIIompA3, yields a sevenfold increase in 2E1 holoenzyme over that obtained from a vector lacking this feature.