Three-step chromatographic purification procedure for the production of a His-tag recombinant kinesin overexpressed in E-coli
Three-step chromatographic purification procedure for the production of a His-tag recombinant kinesin overexpressed in E-coli
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DOI：
10.1016/s0378-4347(99)00524-1
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发表时间：
2000-01-14
期刊：
影响因子：
--
通讯作者：
Santarelli, X
中科院分区：
文献类型：
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作者：
Gibert, S;Bakalara, N;Santarelli, X
利用RT-PCR技术从布氏锥虫（Trypanosomabrucei）中克隆了一个驱动蛋白基因，并在大肠杆菌中高效表达了一个带有组氨酸标签的驱动蛋白。大肠杆菌中，以研究其生化性质，并确定其三维结构的X射线晶体学。从几升培养物开始，使用超声波均化器进行细胞破碎，得到未澄清的原料。然后通过使用膨胀床吸附（EBA）技术（流线螯合）的固定化金属亲和层析（IMAC）从该匀浆中纯化蛋白质。对于该捕获步骤，纯化了100%的重组蛋白，纯度超过90%。该步骤之后进行离子交换色谱法（Q Sepharose Fast Flow）用于中间体纯化（96%纯度，53%回收率），并进行使用Superdex 75的尺寸排阻色谱法作为精制步骤（99%纯度，93%回收率）。然后，我们分离了两种形式的驱动蛋白，二聚体（70%）和单体（30%）。然后可以以快速有效的方式直接从原料中纯化His标签重组蛋白，并分离两种形式的驱动蛋白。(C)2000 Elsevier Science B. V.保留所有权利。
A kinesin gene has been cloned by RT-PCR (reverse transcription polymerase chain reaction) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine-tag) kinesin in E. coli in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography. Starting from several liters of culture, an ultrasonic homogenizer was used for cell disruption and an unclarified feedstock was obtained. From this homogenate, a protein was then purified by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology (Streamline chelating). For this capture step, 100% of the recombinant protein was purified with more than 90% of purity. This step was followed by ion-exchange chromatography (Q Sepharose Fast Flow) for intermediate purification (96% purity, 53% recovery) and by size-exclusion chromatography with Superdex 75 as a polishing step (99% purity, 93% recovery). We then separated two forms of kinesin, a dimer (70%) and a monomer (30%). It was then possible to purify His-tag recombinant protein directly from feedstock in a rapid and efficient way and to isolate two forms of kinesin. (C) 2000 Elsevier Science B.V. All rights reserved.