You J C, McHenry C S
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262.
J Biol Chem. 1993 Aug 5;268(22):16519-27.
The single-stranded nucleocapsid protein that coats the RNA genome of human immunodeficiency virus within the virion core has been produced in Escherichia coli and purified to homogeneity. The mature 55-amino acid protein, normally generated from the gag polyprotein precursor by HIV protease-catalyzed processing of both its amino and carboxyl termini, was produced in E. coli with authentic termini directly, without the need for processing. The protein was purified 30-fold to apparent homogeneity, as determined by both amino acid analysis and SDS-polyacrylamide gel electrophoresis. Sequencing of each terminus of the purified protein indicated that no proteolytic degradation occurred. A molar extinction coefficient (epsilon 280 = 8350 cm-1 M-1) was determined. The purified nucleocapsid protein binds tightly to single-stranded RNA as judged by a nitrocellulose filter binding assay. A binding constant (Kw) of 1 x 10(8) M-1 was calculated. Using fluorescence quenching of nucleocapsid protein upon RNA binding as an assay, a binding site size of seven nucleotides was determined. These results contrast to a larger 15-nucleotide site measured by others for a larger form of nucleocapsid protein-containing sequences from its immature precursor. The possible relevance of these findings are discussed.
包裹在病毒体核心内人类免疫缺陷病毒RNA基因组的单链核衣壳蛋白已在大肠杆菌中产生并纯化至同质状态。成熟的55个氨基酸的蛋白质，通常由HIV蛋白酶催化加工其氨基和羧基末端从gag多蛋白前体产生，在大肠杆菌中直接产生具有真实末端的该蛋白，无需加工。通过氨基酸分析和SDS-聚丙烯酰胺凝胶电泳测定，该蛋白纯化了30倍至表观同质状态。纯化蛋白每个末端的测序表明没有发生蛋白水解降解。测定了摩尔消光系数（ε280 = 8350 cm-1 M-1）。通过硝酸纤维素滤膜结合试验判断，纯化的核衣壳蛋白与单链RNA紧密结合。计算出结合常数（Kw）为1×108 M-1。以RNA结合时核衣壳蛋白的荧光猝灭作为测定方法，确定结合位点大小为7个核苷酸。这些结果与其他人测量的来自其未成熟前体的更大形式的含核衣壳蛋白序列的15个核苷酸的较大位点形成对比。讨论了这些发现的可能相关性。
