Chen S, Court D L
Department of Biochemistry, Fourth Military Medical University, Xi'an, Shaanxi, China.
Chin J Biotechnol. 1991;7(3):177-84.
For high production of Era protein, lambda Ea8.5 gene has been chosen from Gene Bank because the Ea8.5 protein contains a sequence of several amino acids at its N-terminus which is identical to that of Era and also because it can be highly expressed in E. coli. The 5'-end sequence of era gene was substituted by a synthetic oligonucleotide which was identical to that of the 5'-end of Ea8.5 gene, so that the transcripts of era gene was provided with a strong translational initiation signal without changing the amino acid sequence of its translational product. Plasmid pCE31, which harbored the recombined era gene under the control of PL promoter, could express very high levels of Era protein, while synthesis of other cellular proteins was nearly shut off during the period of induction. As a result, Era constituted over 80% of the total cellular protein. Electrophoretic pure Era protein with specific guanine nucleotide-binding activity was obtained by a simple procedure including lyzing the cells and washing the pellet of the lysate.
为了高效生产Era蛋白，已从基因库中选择λEa8.5基因，这是因为Ea8.5蛋白在其N端含有一段与Era相同的氨基酸序列，并且它能在大肠杆菌中高效表达。era基因的5'端序列被一个与Ea8.5基因5'端相同的合成寡核苷酸所取代，这样era基因的转录本就具有了一个强翻译起始信号，而不改变其翻译产物的氨基酸序列。携带重组era基因且受PL启动子控制的质粒pCE31能够表达非常高水平的Era蛋白，而在诱导期间其他细胞蛋白的合成几乎被关闭。结果，Era占细胞总蛋白的80%以上。通过包括裂解细胞和洗涤裂解物沉淀的简单步骤，获得了具有特定鸟嘌呤核苷酸结合活性的电泳纯Era蛋白。
