کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2091061 1081530 2007 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
PCR-based strategy for construction of multi-site-saturation mutagenic expression library
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
PCR-based strategy for construction of multi-site-saturation mutagenic expression library
چکیده انگلیسی

There is an increasing demand for efficient and effective methods to engineer protein variants for industrial applications, structural biology and drug development. We describe a PCR-based strategy that produces multi-site-saturation mutagenic expression library using a circular plasmid carrying the wild-type gene. This restriction digestion- and ligation-independent method involves three steps: 1) synthesis of the degenerate oligonucleotide primers, 2) incorporation of the mutations through PCR, 3) transformation into the expression host. Our strategy is demonstrated through successful construction of an E. coli K12 malic enzyme expression library that contains members with simultaneous mutations on amino acid residues G311, D345 and G397. This method is in principle compatible with any circular vector that can be propagated with a dam+E. coli host to generate protein variant library with multiple changes, including mutation, short sequence deletion and insertion, or any mix of them.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Microbiological Methods - Volume 71, Issue 3, December 2007, Pages 225–230
نویسندگان
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