Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli.
Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli.
复制标题
大肠杆菌硫辛酰胺脱氢酶的过度表达和诱变。
DOI：
10.1042/bj2560741
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
J. Guest
中科院分区：
文献类型：
--
作者：
N. Allison;C. Williams;J. Guest
为了克服尝试突变完整基因时遇到的不稳定问题，开发了一种用于编码大肠杆菌硫辛酰胺脱氢酶的基因过表达和突变的“分裂基因”技术。将硫辛酰胺脱氢酶基因lpd分成两个片段，将其分别亚克隆到M13载体中用于体外诱变，然后在λ PR和λ PL启动子和温度敏感性λ阻遏物的转录控制下在pJLA 504表达载体中重建。热诱导后，E.用携带重建的LPD基因的质粒转化的大肠杆菌细胞含有比野生型生物中正常发现的高4-5倍的硫辛酰胺脱氢酶活性。该策略被用来工程改造的Glu-188-天冬氨酸取代硫辛酰胺脱氢酶，这产生了一种酶具有显着不同的动力学特性。
A 'split-gene' technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene. The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro followed by reconstitution in the pJLA504 expression vector under the transcriptional control of the lambda PR and lambda PL promoters and a temperature-sensitive lambda repressor. After thermo-induction, E. coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism. The strategy was used to engineer a Glu-188----Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties.