کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6490556 | 43375 | 2016 | 30 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A facile and efficient transposon mutagenesis method for generation of multi-codon deletions in protein sequences
ترجمه فارسی عنوان
یک روش موتاژنز ترانسپوزون موثر و کارآمد برای تولید حذف چند کدون در توالی پروتئین
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کلمات کلیدی
ترانزونس نامتقارن، حذف کدون، کتابخانه جهش یافته، پروتئین فلورسنت سبز، خواندن انتخاب قاب،
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 227, 10 June 2016, Pages 27-34
Journal: Journal of Biotechnology - Volume 227, 10 June 2016, Pages 27-34
نویسندگان
Shu-su Liu, Xuan Wei, Qun Ji, Xiu Xin, Biao Jiang, Jia Liu,