کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
10235014 44963 2014 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis
چکیده انگلیسی
Eukaryotic cell-free protein synthesis (CFPS) systems are limited, in part, by inefficient translation initiation. Here, we report three internal ribosome entry site (IRES) sequences from the Dicistroviridae family that are highly active in yeast CFPS. These include the intergenic region (IGR) IRES from cricket paralysis virus (CrPV), plautia stali intestine virus (PSIV) and Solenopsis invicta virus 1 (SINV1). Optimization of combined transcription and translation (Tx/Tl) CFPS reactions primed with linear DNA containing the CrPV IGR IRES resulted in batch synthesis yields of 0.92 ± 0.17 μg/mL luciferase. Further template engineering, such as including the first 12 nt of native CrPV gene, increased yields to 2.33 ± 0.11 μg/mL. We next observed that the inclusion of a 50 nt poly(A) to the 3′ end of the IGR IRES-mediated message increased yields an additional 81% to 4.33 ± 0.37 μg/mL, without any effect on mRNA stability or copy number. This was surprising because the CrPV IGR IRES requires no known translation initiation factors. Lastly, we investigated a method to inhibit background expression through competitive inhibition by supplying the reaction with 5′ cap structure analog. This study highlights the crucial role translation initiation plays in yeast CFPS and offers a simple platform to study IRES sequences.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: New Biotechnology - Volume 31, Issue 5, 25 September 2014, Pages 499-505
نویسندگان
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