Karamyshev A L, Karamysheva Z N, Ito K, Matsufuji S, Nakamura Y
Department of Tumor Biology, Institute of Medical Science, University of Tokyo, Tokyo, 108-8630, Japan.
Biochemistry (Mosc). 1999 Dec;64(12):1391-400.
The polypeptide release factor (eRF1) gene was cloned from rabbit and its overexpression and purification system was established in parallel with that of the eRF1 gene of Tetrahymena thermophila that has been cloned recently in this laboratory. The rabbit eRF1 (Ra-eRF1) is composed of 437 amino acids and is completely identical to human eRF1 though 3% distinct in the nucleotide sequence. This is in sharp contrast to Tetrahymena eRF1 (Tt-eRF1) that is only 57% identical to human eRF1. The recombinant Ra-eRF1 was marked with a histidine tag, overexpressed, and purified to homogeneity by two-step chromatography using Ni-NTA-agarose and Mono Q columns. In contrast to Ra-eRF1, Tt-eRF1 formed aggregates upon overexpression in Escherichia coli, hence it was purified under denaturing conditions, and used to raise rabbit antibody. The resulting anti-Tt-eRF1 antibody proved useful for examining conditions for soluble Tt-eRF1 in test cells. Finally, a soluble Tt-eRF1 fraction was purified from Saccharomyces cerevisiae transformed with the Tt-eRF1 expression plasmid by three steps of affinity and anion exchange chromatography. The cloned Ra-eRF1 gene complemented a temperature-sensitive allele in the eRF1 gene, sup45 (ts), of S. cerevisiae, though the complementation activity was significantly impaired by the histidine tag, whereas Tt-eRF1 failed to complement the sup45 (ts) allele.
从兔中克隆了多肽释放因子（eRF1）基因，并建立了其过表达和纯化系统，同时还建立了本实验室最近克隆的嗜热四膜虫eRF1基因的过表达和纯化系统。兔eRF1（Ra-eRF1）由437个氨基酸组成，与人类eRF1完全相同，尽管核苷酸序列有3%的差异。这与嗜热四膜虫eRF1（Tt-eRF1）形成了鲜明对比，后者与人类eRF1的同源性仅为57%。重组Ra-eRF1带有组氨酸标签，通过镍-亚氨基二乙酸琼脂糖和Mono Q柱两步层析进行过表达并纯化至同质。与Ra-eRF1不同，Tt-eRF1在大肠杆菌中过表达时形成聚集体，因此在变性条件下进行纯化，并用于制备兔抗体。所得的抗Tt-eRF1抗体被证明可用于检测测试细胞中可溶性Tt-eRF1的条件。最后，通过三步亲和和阴离子交换层析从用Tt-eRF1表达质粒转化的酿酒酵母中纯化出可溶性Tt-eRF1组分。克隆的Ra-eRF1基因互补了酿酒酵母eRF1基因sup45（ts）中的一个温度敏感等位基因，尽管组氨酸标签显著损害了互补活性，而Tt-eRF1未能互补sup45（ts）等位基因。
