OVERPRODUCTION AND PURIFICATION OF ESCHERICHIA-COLI TRANSFER RNA-2-GLN AND ITS USE IN CRYSTALLIZATION OF THE GLUTAMINYL-TRANSFER RNA SYNTHETASE-TRANSFER RNA-GLN COMPLEX
OVERPRODUCTION AND PURIFICATION OF ESCHERICHIA-COLI TRANSFER RNA-2-GLN AND ITS USE IN CRYSTALLIZATION OF THE GLUTAMINYL-TRANSFER RNA SYNTHETASE-TRANSFER RNA-GLN COMPLEX
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DOI：
10.1016/0022-2836(88)90524-4
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发表时间：
1988-07-05
影响因子：
5.6
通讯作者：
SOLL, D
中科院分区：
文献类型：
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作者：
PERONA, JJ;SWANSON, R;SOLL, D
我们描述了大肠杆菌 tRNA2Gln 的基因工程过量生产、通过高压液相色谱 (HPLC) 进行的纯化，以及随后用于大肠杆菌谷氨酰胺酰-tRNA 合成酶-tRNAGln 复合物晶体生长的用途。过量生产的 tRNA 占从工程菌株中提取的总 tRNA 的 60% 至 70%。单个阴离子交换 HPLC 柱足以将该同工受体的纯度提高到 90% 至 95%。通过蒸气扩散从含有柠檬酸钠作为沉淀剂的溶液中获得与单体大肠杆菌谷氨酰胺酰-tRNA合成酶复合的该材料的晶体。晶体的衍射范围超过 2.8 .ANG。分辨率 (1 ANG. = 0.1 nm) 和属于正交空间群 C2221，晶胞参数 a = 240.5 ANG.、b = 93.9 ANG.、c = 115.7 ANG.。溶解晶体的凝胶电泳表明蛋白质和 tRNA 均存在。
We describe the genetically engineered overproduction of Escherichia coli tRNA2Gln, its purification by high pressure liquid chromatography (HPLC), and it subsequent use in the growth of crystals of the E. coli glutaminyl-tRNA synthetase-tRNAGln complex. The overproduced tRNA represents 60 to 70% of the total tRNA extracted from the engineered strain. A single anion exchange HPLC column is then sufficient to increase the purity of this isoacceptor to 90 to 95%. Crystals of this material complexed with the monomeric E. coli glutaminyl-tRNA synthetase enzyme were obtained by vapor diffusion from solutions containing sodium citrate as the precipitating agent. The crystals diffract to beyond 2.8 .ANG. resoluation (1 .ANG. = 0.1 nm) and are of the orthorhombic space group C2221 with unit cell parameters a = 240.5 .ANG., b = 93.9 .ANG., c = 115.7 .ANG.. Gel electrophoresis of dissolved crystals demonstrates the presence of both protein and tRNA.