Nalin C M, Purcell R D, Antelman D, Mueller D, Tomchak L, Wegrzynski B, McCarney E, Toome V, Kramer R, Hsu M C
Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110.
Proc Natl Acad Sci U S A. 1990 Oct;87(19):7593-7. doi: 10.1073/pnas.87.19.7593.
Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated. Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis. Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix. Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein. Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons. This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons.
1型人类免疫缺陷病毒的重组Rev蛋白已在大肠杆菌中表达，并通过离子交换和凝胶过滤色谱法进行纯化。已证明纯化后的蛋白与病毒RNA的Rev反应元件具有特异性结合。通过圆二色性和内源荧光光谱对纯化蛋白进行的物理表征表明，该蛋白制剂适用于结构分析。圆二色性测量表明，该蛋白约40-45%为α-螺旋结构。色氨酸荧光测量表明，单一色氨酸残基位于蛋白表面附近。该蛋白的凝胶过滤色谱分析表明，其表观分子量为33,000道尔顿。这表明溶液中的蛋白形成了一个稳定的四聚体，由分子量为13,000道尔顿的单体组成。
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