Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.
Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.
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DOI：
10.1371/journal.pone.0010081
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发表时间：
2010-04-09
期刊：
影响因子：
3.7
通讯作者：
Willbold D
中科院分区：
文献类型：
--
作者：
Glück JM;Hoffmann S;Koenig BW;Willbold D
N-肉豆蔻酰化是许多真核和病毒蛋白的关键共价修饰，由N-肉豆蔻酰基转移酶(NMT)催化。原核生物缺乏内源性NMT活性。通过将异源NMT与目的蛋白共表达，可在大肠杆菌中重组生产N-肉豆蔻酰化蛋白。过去，双质粒系统被用于这一目的。在这里，我们描述了一个单一的载体系统，有效地共表达底物和酶，适合于生产共修饰或翻译后修饰的蛋白质。以HIV-1Nef蛋白为例，验证了该方法的有效性。提出了一种生产高纯度和完全N-肉豆蔻酰化Nef的简单有效的工艺。每升培养基产率约为20 mg肉豆蔻酰化Nef。单一载体策略允许对在大肠杆菌中与异源酶重组共表达的目标蛋白进行不同的修饰。该方法普遍适用，并提供了大量的定量加工的目标蛋白，足以进行全面的生物物理和结构研究。
N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.
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影响因子：
2.9
作者：
AMES, JB;TANAKA, T;IKURA, M
通讯作者：
IKURA, M
DOI：
10.1073/pnas.89.9.4129
发表时间：
1992-05-01
影响因子：
11.1
作者：
DURONIO, RJ;REED, SI;GORDON, JI
通讯作者：
GORDON, JI
影响因子：
3.3
作者：
Foster, John L.;Garcia, J. Victor
通讯作者：
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影响因子：
5.4
作者：
Hermida-Matsumoto, L;Resh, MD
通讯作者：
Resh, MD
影响因子：
8
作者：
Kim, KJ;Kim, HE;Oh, BH
通讯作者：
Oh, BH