Niranjanakumari S, Kurz J C, Fierke C A
Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, NC 27710, USA.
Nucleic Acids Res. 1998 Jul 1;26(13):3090-6. doi: 10.1093/nar/26.13.3090.
Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.
核糖核酸酶P是一种核糖核蛋白复合体，可催化所有前体tRNA分子进行必需的5'端成熟过程。蛋白质组分既能改变RNA组分的构象，又能增强底物亲和力和特异性。为便于进行生化和生物物理研究，使用对大肠杆菌表达进行了优化的最初20个密码子的天然氨基酸序列，在大肠杆菌中过量表达枯草芽孢杆菌核糖核酸酶P（RNase P）的蛋白质组分。开发了一种简单的纯化程序，即在有和没有尿素的情况下使用连续阳离子交换色谱步骤，以纯化大量不含核酸污染的P蛋白。重组蛋白作为RNase P辅因子的身份通过其在低离子强度缓冲液中以1:1化学计量比刺激RNA组分活性的能力得以确立。圆二色性研究表明，P蛋白是α-螺旋和β-折叠二级结构的组合，相当稳定，熔解温度为67℃。所述方法有助于大规模纯化用于高分辨率晶体学分析所需的均一、无RNA的P蛋白，并且可能对制备其他RNA结合蛋白有用。
