Guarino Linda A, Mistretta Toni-Ann, Dong Wen
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Virus Res. 2002 Dec;90(1-2):187-95. doi: 10.1016/s0168-1702(02)00152-1.
PP31 is a baculovirus protein that is essential for viral late gene expression. To study the role of PP31 in late transcription in vitro, it was purified from infected insect cells. A combination of heparin affinity, cation exchange chromatography, and gel filtration was used to purify native non-tagged protein. Nearly 5 mg of PP31 was obtained from 95 mg of nuclear extract confirming that PP31 is an abundant viral protein. DNA binding assays revealed that PP31 binds to single-stranded and double-stranded DNA with equal affinities. Addition of PP31 to in vitro transcription assays with purified baculovirus RNA polymerase resulted in a strong inhibition of transcription. This indicates that the viral RNA polymerase was not able to displace PP31, and suggests that other late expression factors may function to help RNA polymerase bind to PP31-coated templates.
PP31是一种杆状病毒蛋白，对病毒晚期基因表达至关重要。为了在体外研究PP31在晚期转录中的作用，它是从受感染的昆虫细胞中纯化得到的。采用肝素亲和、阳离子交换色谱和凝胶过滤相结合的方法来纯化天然无标签蛋白。从95毫克核提取物中获得了近5毫克PP31，证实PP31是一种丰富的病毒蛋白。DNA结合试验表明，PP31以相等的亲和力结合单链和双链DNA。将PP31添加到用纯化的杆状病毒RNA聚合酶进行的体外转录试验中，导致转录受到强烈抑制。这表明病毒RNA聚合酶无法取代PP31，并提示其他晚期表达因子可能发挥作用，帮助RNA聚合酶结合到被PP31包被的模板上。
