کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2185903 1096023 2010 14 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیولوژی سلول
پیش نمایش صفحه اول مقاله
An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli
چکیده انگلیسی

We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNACUAopt in a single-vector construct. The optimized suppressor tRNACUAopt afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Molecular Biology - Volume 395, Issue 2, 15 January 2010, Pages 361–374
نویسندگان
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