Henry K A, Zwieb C, Fried H M
Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, 27599, USA.
Protein Expr Purif. 1997 Feb;9(1):15-26. doi: 10.1006/prep.1996.0667.
The signal recognition particle (SRP) is a ribonucleoprotein complex that mediates translocation of proteins into the endoplasmic reticulum. Protein SRP19 is an essential structural component of SRP and is believed to promote assembly of the particle. In order to have a convenient source for the purification of milligram amounts of SRP19, we expressed in Escherichia coli a human SRP19 cDNA with an amino-terminal addition of six histidine residues. Expression at 25 degrees C eliminated formation of insoluble SRP19 and resulted in accumulation of soluble hexahistidine-SRP19 to 68% of total cell protein after 24 h. Metal chelation chromatography yielded 40 mg of hexahistidine-SRP19 per liter of culture, with a purity slightly greater than 97%. To examine protein function, the RNA-binding properties of the purified protein were determined by RNA electromobility shift assays. The histidine-tagged SRP19 bound specifically to a 150-nucleotide RNA derived from SRP RNA, with an apparent Kd of 1 nM, and bound, with greatly reduced affinity, to a mutagenized form of the SRP RNA derivative that contained an altered helix 6 tetranucleotide loop. The purified protein was also photochemically crosslinked to the 150-nucleotide SRP RNA fragment, providing the means to potentially identify portions of hexahistidine-SRP19 which are in close proximity to the RNA molecule.
信号识别颗粒（SRP）是一种核糖核蛋白复合体，介导蛋白质向内质网的转运。蛋白质SRP19是SRP的一种必需结构成分，被认为能促进该颗粒的组装。为了获得一个方便的来源以纯化毫克量的SRP19，我们在大肠杆菌中表达了一个在氨基末端添加了六个组氨酸残基的人SRP19 cDNA。在25℃下表达可消除不溶性SRP19的形成，并在24小时后使可溶性六组氨酸 - SRP19积累至总细胞蛋白的68%。金属螯合层析每升培养物可产生40毫克六组氨酸 - SRP19，纯度略高于97%。为了检测蛋白质功能，通过RNA电泳迁移率变动分析来确定纯化蛋白质的RNA结合特性。带有组氨酸标签的SRP19特异性结合源自SRP RNA的150个核苷酸的RNA，其表观解离常数（Kd）为1 nM，并以大大降低的亲和力结合到含有改变的螺旋6四核苷酸环的SRP RNA衍生物的诱变形式上。纯化的蛋白质还通过光化学交联到150个核苷酸的SRP RNA片段上，这为潜在地鉴定六组氨酸 - SRP19中与RNA分子紧密相邻的部分提供了手段。
