A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons
A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons
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DOI：
10.1093/protein/gzr060
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发表时间：
2012-02-01
影响因子：
2.4
通讯作者：
Cropp, T. Ashton
中科院分区：
文献类型：
--
作者：
Liu, Jia;Cropp, T. Ashton
随机诱变后进行选择或筛选是一种常用的改善蛋白质功能的策略。尽管有许多可用的随机诱变方法，但几乎所有方法都在核苷酸水平上产生突变。理想的诱变方法将允许产生“密码子突变”，以改变具有所选的确定或混合氨基酸的蛋白质序列。在此，我们报告了一种允许一个、两个或三个连续密码子突变的方法。该方法的关键是开发具有不对称末端序列的Mu转座子变体。作为该方法的示范，我们对编码超折叠GFP（sfGFP）的基因进行了多密码子扫描。从每个文库中随机选择的50个克隆的表征表明，这三个文库中超过40%的突变体含有具有低位点偏好的无缝框内突变。通过从每个文库中筛选仅500个菌落，我们成功地鉴定了几个光谱偏移突变，包括发现在UV区域具有单个激发峰的S205D变体。
Random mutagenesis followed by selection or screening is a commonly used strategy to improve protein function. Despite many available methods for random mutagenesis, nearly all generate mutations at the nucleotide level. An ideal mutagenesis method would allow for the generation of 'codon mutations' to change protein sequence with defined or mixed amino acids of choice. Herein we report a method that allows for mutations of one, two or three consecutive codons. Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences. As a demonstration of the method, we performed multi-codon scanning on the gene encoding superfolder GFP (sfGFP). Characterization of 50 randomly chosen clones from each library showed that more than 40% of the mutants in these three libraries contained seamless, in-frame mutations with low site preference. By screening only 500 colonies from each library, we successfully identified several spectra-shift mutations, including a S205D variant that was found to bear a single excitation peak in the UV region.