Creation of genome-wide protein expression libraries using random activation of gene expression
Creation of genome-wide protein expression libraries using random activation of gene expression
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DOI：
10.1038/88107
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发表时间：
2001-05-01
影响因子：
46.9
通讯作者：
Ducar, M
中科院分区：
文献类型：
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作者：
Harrington, JJ;Sherf, B;Ducar, M
在这里，我们报告使用随机激活基因表达（random activation of gene expression，缩写为EMPs）来创建全基因组蛋白质表达文库。发现仅含有5 × 10（6）个单独克隆的cDNA文库表达每个测试基因，包括在亲本细胞系中通常沉默的基因。此外，内源性基因以相似的频率被激活，并在从多种人细胞系创建的cDNA文库中以相似的水平表达，这表明cDNA文库是固有地标准化的。使用大量的cDNA克隆来分离19，547个人类基因簇，当与表达序列标签（EST）和互补DNA（cDNA）的公共数据库进行测试时，其中53%是新的。单个克隆的分离证实，活化的内源基因可以高水平表达以产生生物活性蛋白。cDNA文库和cDNA表达克隆的性质非常适合于许多生物技术应用，包括基因发现、蛋白质表征、药物开发和蛋白质制造。
Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, similar to 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.