A set of ligation-independent in vitro translation vectors for eukaryotic protein production
A set of ligation-independent in vitro translation vectors for eukaryotic protein production
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DOI：
10.1186/1472-6750-8-32
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发表时间：
2008-03-27
影响因子：
3.5
通讯作者：
Meszaros, Tamas
中科院分区：
文献类型：
--
作者：
Bardoczy, Viola;Geczi, Viktoria;Meszaros, Tamas
背景：过去十年带来了蛋白质研究的复兴，加速了蛋白质组学各方面高通量方法的发展。目前，大多数蛋白质合成系统利用活细胞翻译蛋白质的能力，但它们的应用受到几个因素的限制。一种更灵活的替代蛋白质生产方法是体外无细胞蛋白质翻译。目前可用的体外翻译系统适用于高通量机器人蛋白质生产，满足蛋白质组学研究的要求。基于小麦胚芽提取物的体外翻译系统可能是最有前途的方法，因为许多真核蛋白可以以其天然折叠形式低成本高效地合成。虽然目前可用的小麦胚体外翻译系统的载体保证了高生产力，但它们不符合最先进的蛋白质组学的要求。目标基因必须使用限制性内切酶插入，质粒不编码可切割的亲和纯化标签。结果：设计了4个基于体外蛋白翻译的小麦胚芽提取物结扎独立克隆（LIC）载体。在这些构建体中，RNA转录由T7或SP6噬菌体聚合酶驱动，并且可以添加两个TEV蛋白酶可切割亲和力标签来辅助蛋白质纯化。为了评估我们改进的载体，在所有四个构建体中克隆了一个植物有丝分裂原激活蛋白激酶。对该真核蛋白激酶的纯化表明，所有构建体的功能都符合预期：LIC插入PCR片段有效，GST-Sepharose或MagneHis颗粒对翻译蛋白进行亲和纯化，获得高纯度的激酶，并且在不同的反应条件下都可以有效地去除亲和标签。此外，高的体外激酶活性证明了纯化蛋白的正确折叠。结论：构建了4个新的体外翻译载体，可实现快速、平行克隆和蛋白纯化，是高通量生产真核蛋白的有效分子工具。
Background: The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags.Results: We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein.Conclusion: Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.