Brown Adrian, Affleck Val, Kroon Johan, Slabas Antoni
School of Biological and Biomedical Sciences, Science Laboratories, University of Durham, South Road, Durham DH1 3LE, UK.
FEBS Lett. 2009 Jan 22;583(2):363-8. doi: 10.1016/j.febslet.2008.12.022. Epub 2008 Dec 25.
The predicted mature portion of a putative 3-hydroxyacyl-ACP dehydratase (DH) from Arabidopsis was linked to an N-terminal poly-histidine-tag and the fusion protein expressed in Escherichia coli. Soluble dehydratase was present on induction at 25 degrees C and pure dehydratase eluted from a nickel-affinity column in 0.2-0.5M imidazole. High concentrations of imidazole were necessary to retain enzyme solubility. The dehydratase reaction is reversible and 3-hydroxybutyryl- and 2-butenoyl-ACP substrates were prepared from E. coli apo-ACP. Analysis of these suggested contamination of apo-ACP with dehydratase and an additional reverse-phase chromatographic step was required during acyl carrier protein (ACP) preparation. Activity of purified dehydratase was demonstrated by mass spectrometry using 2-butenoyl-ACP, providing the first functional experimental evidence for plant DH gene sequences.
来自拟南芥的一种假定的3-羟酰基-ACP脱水酶（DH）的预测成熟部分与N端多组氨酸标签相连，并在大肠杆菌中表达融合蛋白。在25℃诱导时存在可溶性脱水酶，纯脱水酶在0.2 - 0.5M咪唑中从镍亲和柱上洗脱。需要高浓度的咪唑来保持酶的溶解性。脱水酶反应是可逆的，3-羟丁酰基-和2-丁烯酰基-ACP底物由大肠杆菌脱辅基-ACP制备。对这些的分析表明脱辅基-ACP被脱水酶污染，在酰基载体蛋白（ACP）制备过程中需要额外的反相色谱步骤。使用2-丁烯酰基-ACP通过质谱法证明了纯化脱水酶的活性，为植物DH基因序列提供了首个功能性实验证据。
