Measuring the activity of protein variants on a large scale using deep mutational scanning.
Measuring the activity of protein variants on a large scale using deep mutational scanning.
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DOI：
10.1038/nprot.2014.153
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发表时间：
2014-09
期刊：
影响因子：
14.8
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中科院分区：
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深度突变扫描将蛋白质功能选择与高通量DNA测序相结合，以便大规模量化蛋白质变体的活性。首先，对感兴趣的蛋白质功能进行合适的选择系统的鉴定和验证。其次，创建一个变体库，将其引入选择系统并进行选择。第三，在整个选择过程中恢复文库DNA并进行深度测序。最后，根据选择过程中变体出现频率的变化计算每个变体的功能分数。该方案描述了必须执行的步骤，以生成由多达数十万个感兴趣的蛋白质变体的功能评分组成的大规模诱变数据集。建立测定、生成变异文库、进行选择及其伴随的测序大约需要4-6周；初步数据分析可在1周内完成。
Deep mutational scanning marries selection for protein function to high-throughput DNA sequencing in order to quantify the activity of variants of a protein on a massive scale. First, an appropriate selection system for the protein function of interest is identified and validated. Second, a library of variants is created, introduced into the selection system and subjected to selection. Third, library DNA is recovered throughout the selection and deeply sequenced. Finally, a functional score for each variant is calculated based on the change in the frequency of the variant during the selection. This protocol describes the steps that must be carried out to generate a large-scale mutagenesis data set consisting of functional scores for up to hundreds of thousands of variants of a protein of interest. Establishing an assay, generating a library of variants, and carrying out a selection and its accompanying sequencing takes on the order of 4–6 weeks; the initial data analysis can be completed in 1 week.