Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules
Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules
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DOI：
10.1006/abio.2001.5182
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发表时间：
2001-08-15
影响因子：
2.9
通讯作者：
Cass, AEG
中科院分区：
文献类型：
--
作者：
Halliwell, CM;Morgan, G;Cass, AEG
在嗜热脂肪芽孢杆菌的L-乳酸脱氢酶（LDH）的N-或C-末端融合一个（多）组氨酸标签以促进这些酶的纯化和固定化。与野生型和N-末端标记的变体相比，C-末端标记的酶显示出较低的活性。通过在5 ′-AMP-琼脂糖树脂上的酶亲和层析和通过尺寸排阻层析研究了这种活性损失的原因。C-末端标记的酶可以被分离成无活性的未结合部分和活性的结合部分。在三种酶的尺寸排阻色谱上观察到C-末端标记的酶与N-末端标记的和野生型LDH之间的进一步差异。这些数据表明，在C-末端引入“组氨酸标签”可能会诱导LDH的错误折叠，并作为一个警告，（多）组氨酸标签的引入可能会产生不可预见的蛋白质的变化。(C)北京：科学出版社.
A (poly)histidine tag was fused to either the N- or the C-terminus Of L-lactate dehydrogenase (LDH) of Bacillus stearothermophilus to facilitate purification and immobilization of these enzymes. The C-terminally tagged enzyme displayed lower activity compared both to the wild-type and to the N-terminally tagged variant. The reason for this loss of activity was investigated by affinity chromatography of the enzymes on a 5 ' -AMP-Sepharose resin and by size-exclusion chromatography. The C-terminally tagged enzyme could be separated into an inactive, unbound fraction and an active, bound fraction. Further differences between the C-terminally tagged enzyme and the N-terminally tagged and wild-type LDH were observed on size-exclusion chromatography of the three enzymes. These data suggest that the introduction of a "his-tag" at the C-terminus may induce misfolding of the LDH and serve as a warning that the introduction of a (poly)histidine tag can produce unforseen changes in a protein. (C) 2001 Academic Press.