Stammers D K, Ross C K, Idriss H, Lowe D M
Department of Molecular Sciences, Wellcome Research Laboratories, Beckenham, Kent, England.
Eur J Biochem. 1992 Jun 1;206(2):437-40. doi: 10.1111/j.1432-1033.1992.tb16944.x.
Human-immunodeficiency-virus-type-1 reverse transcriptase exists in virions as a heterodimer of a M(r) 66,000 subunit and its C-terminally truncated form of M(r) 51,000, but, when expressed as a recombinant M(r) 66,000 protein, a mixture of heterodimers and homodimers results which co-purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified M(r) 66,000 and 51,000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the M(r) 66,000 protein. Heterodimers having greater asymmetry have also been prepared by recombining Mr 66,000 subunits containing single-point or deletion mutations, with wild-type M(r) 51,000 subunits, and the resulting heterodimers analysed.
1型人类免疫缺陷病毒逆转录酶以一种由分子量为66,000的亚基及其C末端截短形式（分子量为51,000）组成的异二聚体存在于病毒粒子中，但是，当作为重组的分子量为66,000的蛋白质表达时，会产生异二聚体和同二聚体的混合物，这些混合物通过大多数常规技术共同纯化。我们描述了一种疏水色谱法，该方法能对这两种形式的蛋白质进行基线分离。此方法已用于纯化由分别表达和纯化的分子量为66,000和51,000的亚基重组形成的异二聚体，从而得到更为均一的异二聚体制剂。重组后的异二聚体与标准异二聚体表现出相似的动力学特性和核糖核酸酶H活性，且比活性显著高于原始的分子量为66,000的蛋白质同二聚体。通过将含有单点或缺失突变的分子量为66,000的亚基与野生型分子量为51,000的亚基重组，也制备出了具有更大不对称性的异二聚体，并对所得异二聚体进行了分析。
