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首页> 外文期刊>Cell cycle >Codon-optimized cloning, expression and characertization of the C-terminal region of human apoptotic protein GADD34 in Escherichia coli.
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Codon-optimized cloning, expression and characertization of the C-terminal region of human apoptotic protein GADD34 in Escherichia coli.

机译：CORCHERICHIA COLI中人类凋亡蛋白GADD34的C末端区域的密码子优化克隆，表达和致尖。

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摘要

The human GADD34 (Growth Arrest and DNA Damage-inducible 34) is the product of an apoptosis- and DNA-damage-inducible gene. The C-terminus domain of GADD34 is highly homologous to HSV-1 gamma-1 34.5, HSV-2 and the African swine fever virus virulence-associated factor NL-S. Among these viral proteins, HSV-1 gamma 34.5 protein is known to prevent apoptosis of viral-infected cells. Because of the difficulty in expressing GADD34 protein or any of its fragments, including the C-terminus (amino acids 533-632) in E. coli, partially due to sub-optimal expression of eukaryotic codons in prokaryotic E. coli, we used a codon-optimized cloning scheme to construct the eukaryotic gene that codes for GADD34(533-632). We derived a novel PCR protocol to assemble 20 oligonucleotides into the synthetic GADD34(533-632) gene. The clear advantage of using this protocol is that the assembled gene is without the mutation and deletion that are usually of a major problem in constructing synthetic genes. The synthetic GADD34(533-632) gene was cloned, expressed, and purified in large quantity. We obtained approximately 50 mg of GADD34(533-632) protein per liter minimum-medium culture. To our knowledge, this is the first report of a large-scale production of the C-terminus of GADD34. The production and purification of GADD34(533-632) in large quantity are essential for structure determination as well as for understanding its interactions with other proteins such as phosphatase 1-alpha using NMR spectroscopy and other biophysical methods.
机译：人的Gadd34（生长停滞和DNA损伤诱导34）是凋亡和DNA损伤诱导基因的产物。 GADD34的C-末端结构域对HSV-1γ-1 34.5，HSV-2和非洲猪瘟病毒毒力相关因子NL-S高度同源。在这些病毒蛋白中，已知HSV-1γ34.5蛋白是预防病毒感染细胞的凋亡。由于难以表达Gadd34蛋白质或任何片段，包括大肠杆菌中的C-末端（氨基酸533-632），部分原因是由于原核大肠杆菌中真核密码子的次良好表达，我们使用了密码子优化的克隆方案，以构建用于GADD34（533-632）的核查的真核基因。我们衍生一种新的PCR方案，将20个寡核苷酸组合成合成的GADD34（533-632）基因。使用该方案的明显优点是组装基因没有突变和缺失，通常是构建合成基因的主要问题。克隆，表达并纯化合成的GADD34（533-632）基因。我们获得了约50毫克的每升最小培养基培养物的Gadd34（533-632）蛋白质。为我们的知识，这是第一个关于GADD34的C-TOMPINUS的大规模生产的第一个报告。 Gadd34（533-632）大量的生产和纯化对于结构测定至关重要，以及使用NMR光谱和其他生物物理方法理解其与磷酸酶1-α的其他蛋白质的相互作用。

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