title: A facile and efficient transposon mutagenesis method for generation of multi-codon deletions in protein sequences.
abstract: 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.
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. Electronic address: liushs@shanghaitech.edu.cn.
authors.forename: Shu-Su
authors.initials: SS
authors.lastname: Liu
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. Electronic address: weixuan@shanghaitech.edu.cn.
authors.forename: Xuan
authors.initials: X
authors.lastname: Wei
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. Electronic address: jiqun@shanghaitech.edu.cn.
authors.forename: Qun
authors.initials: Q
authors.lastname: Ji
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. Electronic address: xinxiu@shanghaitech.edu.cn.
authors.forename: Xiu
authors.initials: X
authors.lastname: Xin
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. Electronic address: jiangbiao@shanghaitech.edu.cn.
authors.forename: Biao
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA. Electronic address: liujia@shanghaitech.edu.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017384:Sequence Deletion / Q000235:genetics*
chemical_list: D003062:Codon; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins
keywords: Asymmetric transposon; Codon deletions; Green fluorescent protein; Mutant library; Reading frame selection