首页> 外文期刊>Plant Science: An International Journal of Experimental Plant Biology >Evaluation of genetically attached histidine affinity tails for purification of lactate dehydrogenase from transgenic tobacco.
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Evaluation of genetically attached histidine affinity tails for purification of lactate dehydrogenase from transgenic tobacco.

机译:用于从转基因烟草中纯化乳酸脱氢酶的遗传附着组氨酸亲和尾的评估。

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摘要

Lactate dehydrogenase (LDH) from Bacillus stearothermophilus has been expressed in transgenic tobacco. To facilitate purification, polyhistidine tails were fused to the 5'-end of the gene. Two different tails, His6 and His-X3-His-X3-His, were compared regarding their effect on LDH gene expression and metal ion specificity. His6 exhibited strong binding to all of the tested transition metals (Zn2+, Co2+, Ni2+ and Cu2+) while the alpha-helical His-X3-His-X3-His showed a preference for Co2+ over Zn2+.This alpha-helical His tail also increased the level of gene expression compared to the native enzyme construct. The histidine modified proteins could be successfully purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn2+,Co2+ or Ni2+. LDHHis6 could also be precipitated from a crude tobacco protein extract using ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) charged with Zn2+.
机译:来自嗜热脂肪芽孢杆菌的乳酸脱氢酶 (LDH) 已在转基因烟草中表达。为了便于纯化,将多组氨酸尾部融合到基因的 5' 端。比较了两种不同的尾巴,His6 和 His-X3-His-X3-His,它们对 LDH 基因表达和金属离子特异性的影响。His6与所有测试的过渡金属(Zn2+、Co2+、Ni2+和Cu2+)表现出较强的结合力,而α-螺旋His-X3-His-X3-His表现出对Co2+的偏好,而不是Zn2+。与天然酶构建体相比,这种α-螺旋His尾巴也增加了基因表达水平。组氨酸修饰蛋白可在装有Zn2+、Co2+或Ni2+的固定化金属亲和层析(IMAC)色谱柱上成功纯化。LDHHis6 也可以使用带有 Zn2+ 的乙二醇-双(β-氨基乙基醚)N,N,N',N'-四乙酸 (EGTA) 从粗烟草蛋白提取物中沉淀。

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