Attempts to increase the expression of rat liver mitochondrial aldehyde dehydrogenase in E. coli by altering the mRNA.
Attempts to increase the expression of rat liver mitochondrial aldehyde dehydrogenase in E. coli by altering the mRNA.
复制标题
尝试通过改变 mRNA 来增加大肠杆菌中大鼠肝线粒体醛脱氢酶的表达。
DOI：
10.1007/978-1-4615-2904-0_9
复制
发表时间：
1993
影响因子：
--
通讯作者：
Weiner,H
中科院分区：
文献类型：
--
作者：
Zheng,CF;Weiner,H
我们最近克隆并表达了大鼠和人肝线粒体第二类乙醛脱氢酶的编码基因(FarReset等人，1989；Jeng和Weiner，1991，郑等人，提交)。重组表达的酶与天然酶的性质几乎相同。有了表达系统，我们就可以进行定点突变来研究氨基酸残基的作用，例如谷氨酸487、组氨酸235、半胱氨酸302和丝氨酸74，仅举几例。虽然我们可以用传统的方法(Jeng和Weiner，1991)或羟基苯乙酮-琼脂糖亲和矩阵(Ghenbot和Weiner提交)来分离该酶，但产酶量非常低。用pt7-7表达的蛋白质中，有多达20%来自它们的cDNA(参见Marston，1986)，而我们用同样的系统，每升培养细胞只能获得1-2毫克的ALDH。为了研究重组表达的酶的化学和物理性质，我们有必要生产更多的酶。
We have recently cloned and expressed the cDNA coding for rat and human liver mitochondrial class 2 aldehyde dehydrogenase (Farreset al., 1989; Jeng and Weiner, 1991, Zhenget al., submitted). The recombinantly expressed enzymes were found to have properties virtually identical to those of the native enzyme. Having an expression system has allowed us to perform site directed mutagenesis to study the role of amino acid residues such as glutamate 487, histidine 235, cysteine 302, and serine 74, to mention just a few. Though we could isolate the enzyme using conventional procedures (Jeng and Weiner, 1991) or a hydroxyacetophenone-Sepharose affinity matrix (Ghenbot and Weiner, submitted) the amount of enzyme produced was very low. Using the plasmid pT7–7 others have reported that as much as 20% of the protein expressed was from their cDNA (see Marston, 1986 for a review) while we could obtain just 1–2 mg of ALDH per liter of cultured cells using the same system. In order for us to study chemical and physical properties of the recombinantly expressed enzymes it will be necessary for us to produce much more enzyme.