突变
遗传学
转座子突变
生物
转座因子
转座酶
终止密码子
索引
突变
起始密码子
蛋白质工程
计算生物学
定向诱变
基因
突变体
基序列
生物化学
基因型
单核苷酸多态性
酶
作者
Shu-su Liu,Wei Xuan,Qun Ji,Xiaoyi Xin,Biao Jiang,Jia Liu
标识
DOI:10.1016/j.jbiotec.2016.03.038
摘要
Substitutions, insertions and deletions are all important mutation events in natural and laboratory protein evolution. However, protein engineering using insertions and deletions (indels) is hindered by the lack of a convenient mutagenesis method. Here, we describe a general transposon mutagenesis method that allows for removal of up to five consecutive in-frame codons from a random position of a target protein. This method, referred to as codon deletion mutagenesis (CDM), relies on an engineered Mu transposon that carries asymmetric terminal sequences flanking the MuA transposase recognition sites. CDM requires minimal DNA manipulations, and can generate multi-codon deletions with high efficiency (>90%). As a proof of principle, we constructed five libraries of green fluorescent protein (GFP) containing one to five random codon deletions, respectively. Several variants with multi-codon deletions remained fluorescent, none of which could be easily identified using traditional mutagenesis method. CDM provides a facile and efficient approach to sampling a protein sequence with multi-codon deletions. It will not only facilitate our understanding of the effects of amino acid deletions on protein function but also expedite protein engineering using deletion mutagenesis.
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