与大肠杆菌碱性磷酸酶融合的化学合成α-新内啡肽基因的表达

Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase.

作者信息

Ohsuye K, Nomura M, Tanaka S, Kubota I, Nakazato H, Shinagawa H, Nakata A, Noguchi T

出版信息

Nucleic Acids Res. 1983 Mar 11;11(5):1283-94. doi: 10.1093/nar/11.5.1283.

DOI:10.1093/nar/11.5.1283

PMID:6298740

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC325796/

Abstract

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.

摘要

我们之前通过化学合成并插入到pK013质粒的大肠杆菌β-半乳糖苷酶基因中的α-新内啡肽（α-NE）基因，已被切除并与大肠杆菌碱性磷酸酶（APase）基因融合。其中一个转化体被命名为E15/pAαNE1。在APase基因调控下，APase-α-NE嵌合蛋白以每个细胞1.3×10⁶个分子的水平表达，约占细胞总蛋白的60%。经溴化氰处理的E15/pAαNE1的高效液相色谱图谱非常简单，这反映出嵌合蛋白含量高且其中甲硫氨酸残基数量少。一系列编码APase-α-NE嵌合蛋白的基因被克隆到大肠杆菌中，这些嵌合蛋白的30至94个C末端氨基酸残基被（甲硫氨酸）-α-NE取代。对嵌合蛋白向周质空间的转运进行了研究。所有嵌合蛋白显然都被信号肽酶加工处理，但很少（如果有的话）被转运到周质空间。