Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 tat protein
Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 tat protein
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DOI：
10.1006/prep.1996.0076
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发表时间：
1996-08-01
影响因子：
1.6
通讯作者：
Rosch, P
中科院分区：
文献类型：
--
作者：
Kirsch, T;Boehm, M;Rosch, P
我们建立了全长HIV-1反式激活因子（达特）蛋白在大肠杆菌中的表达系统。通过构建用于在肠道细菌中高水平表达的合成基因，可以高产率获得重组蛋白。达特序列与N-末端组氨酸标签的融合允许通过单个色谱步骤快速纯化融合蛋白。用CNBr切割融合蛋白后，可通过使用MonoS柱获得纯达特。用三（2-羧乙基）膦- HCl还原蛋白质并随后逐步重折叠产生具有生物活性的达特。通过质谱法证明样品纯度和蛋白质质量与氨基酸序列预期质量的同一性。核磁共振光谱显示细菌表达的和化学合成的达特蛋白的同一性（P. Bayer等人，1995，J. Mol. Biol. 247，529-535）。将达特基因在E.大肠杆菌使同位素标记成为实验的先决条件，以阐明Tat-反式激活反应元件复合物的结构。(C)出版社：Academic Press，Inc.
We have established an expression system for full-length HlV-1 transactivator (Tat) protein in Escherichia coli. By constructing a synthetic gene for high level expression in enteric bacteria, the recombinant protein can be obtained in high yield. Fusion of the Tat sequence to an N-terminal histidine tag allows the rapid purification of the fusion protein through a single chromatographic step. After cleavage of the fusion protein with CNBr, pure Tat can be obtained through the use of a MonoS column. Reduction of the protein with Tris (2-carboxyethyl)phosphine - HCl and subsequent stepwise refolding yields biologically active Tat. Sample purity and the identity of the protein mass with the mass expected from the; amino acid sequence was demonstrated by mass spectrometry. Nuclear magnetic resonance spectroscopy showed the identity of bacterially expressed and chemically synthesized Tat protein (P. Bayer et al., 1995, J. Mol. Biol. 247, 529-535). The expression of Tat in E. coli enables isotope labeling as a prerequisite for experiments toward the elucidation of the structure of the Tat-trans-activation response element complex. (C) 1996 Academic Press, Inc.