Existence of β-methylnorleucine in recombinant hirudin produced by Escherichia coli
Existence of β-methylnorleucine in recombinant hirudin produced by Escherichia coli
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DOI：
10.1016/s0168-1656(01)00396-0
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发表时间：
2002-02-14
影响因子：
4.1
通讯作者：
Hayashi, H
中科院分区：
文献类型：
--
作者：
Muramatsu, R;Negishi, T;Hayashi, H
水蛭素是一种有效的凝血酶抑制剂，其编码基因在大肠杆菌中表达，大肠杆菌是最广泛使用的宿主。当重组水蛭素类似物CX-397被E.大肠杆菌（600 mg L（-1））中，在培养基中不存在营养氨基酸的情况下，在终产物中观察到两种衍生物的存在，在反相高效液相色谱上具有极大增加的保留时间。每种衍生物是由于CX-397中Ile 29或Ile 59处的异亮氨酸残基的甲基化。其结构可推断为β-甲基正亮氨酸（betaMeNle. (2S 3S）-2-氨基-3-甲基己酸）。β-MeNle的修饰途径不被认为是蛋白质的翻译后修饰，因为Ile在其侧链中没有功能基团。此外，β-MeNle是由与E. coli（J. Antibiot. 34（1981 a）1278）。这些结果表明，培养基中缺乏营养氨基酸导致β-MeNle在E. coli中，然后用E. coli异亮氨酰-tRNA合成酶，并掺入过量生产的重组蛋白中。(C)2002 Elsevier Science B. V.保留所有权利。
A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli, which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l(-1)) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as beta-methylnorleucine (betaMeNle. (2S,3S)-2-amino-3-methylhexanoic acid). The modification pathway of betaMeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain, Additionally, betaMeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of betaMeNle in E. coli, which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein. (C) 2002 Elsevier Science B.V. All rights reserved.