Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes
Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes
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DOI：
10.1016/j.pep.2007.10.018
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发表时间：
2008-02-01
影响因子：
1.6
通讯作者：
Martinez-Yamout, Maria
中科院分区：
文献类型：
--
作者：
Sugase, Kenji;Landes, Mindy A.;Martinez-Yamout, Maria
翻译后修饰在真核细胞信号转导中起着重要作用。为了阐明具有特定翻译后修饰的蛋白质的生物学功能，有必要分离修饰的蛋白质。然而，特别是在大规模制备中，难以将修饰的氨基酸掺入蛋白质的特定位置。为了在大肠杆菌（Escherichia coli，E. coli），构建了含有蛋白和相应酶基因的共表达载体。该蛋白和酶在同一个E.大肠杆菌细胞，并在体内对蛋白质进行后修饰。利用该系统，在大肠杆菌中，转录激活因子环AMP反应元件结合蛋白（CREB）在Ser-133处被磷酸化，缺氧诱导因子-1 α（HIF-1 α）在Asn-803处被羟基化。杆菌虽然我们使用的蛋白质结构非常灵活，并且容易被E.在大肠杆菌中单独表达时，将链球菌G蛋白（GB 1）的B1结构域融合到蛋白的N-末端，显著提高了产量。通过基质辅助激光解吸/电离飞行时间（MALDI-TOF）和NMR证实CREB的位点特异性磷酸化和HIF-1 α的羟基化。我们的GB 1-融合共表达系统可以以与常规蛋白表达相同的方式在E.大肠杆菌，使其成为一种灵活和经济的方法来生产大量的后修饰的蛋白质。(C)2007爱思唯尔公司All rights reserved.
Post-translational modification plays crucial roles in signal transduction in eukaryotic cells. To elucidate the biological function of a protein with a specific post-translational modification, it is necessary to isolate the modified protein. However, it is difficult to incorporate a modified amino acid into a specific position of a protein, in particular, in a large-scale preparation. In order to prepare post-translationally modified proteins in Escherichia coli (E. coli), we have constructed co-expression vectors that contain protein and corresponding enzyme genes. The protein and enzyme are co-expressed in the same E. coli cells and the protein is post-translationally modified in vivo. By using this system, the transcriptional activator cyclic-AMP-response-element-binding protein (CREB) was phosphorylated at Ser-133 and the hypoxia-inducible factor-1 alpha (HIF-1 alpha) was hydroxylated at Asn-803 in E. coli. Although the constructs of the proteins we used are very flexible and susceptible to degradation by proteases in E. coli when they are expressed alone, the B1 domain of streptococcal protein G (GB1) fused to the N-terminus of the proteins increased the yields dramatically. Site-specific phosphorylation of CREB and hydroxylation of HIF-1 alpha were confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and NMR. Our GB1-fusion co-expression system can be used in the same way as conventional protein expression in E. coli, making it a flexible and economical method to produce a large amount of a post-translationally modified protein. (C) 2007 Elsevier Inc. All rights reserved.