کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
21020 43202 2011 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Firm wheat-germ cell-free system with extended vector usage for high-throughput protein screening
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Firm wheat-germ cell-free system with extended vector usage for high-throughput protein screening
چکیده انگلیسی

ABSTRACTThe wheat germ cell-free system is composed out of five basic steps, growth of Escherichia coli harboring plasmid, first colony-PCR, second PCR, transcription, and translation. Improvements of culture medium, colony based PCR, and modifications within the split primer set of the second PCR amplify both DNA and RNA levels. This yields more than 5 times increase in protein amount for pEU-originated templates. Especially, for the low PCR-amplifiable vectors with pET-origin, it leads to 30 fold higher product amount in translation. This broadens the range of usable vectors, overcoming the existing cell-free system limitations for high-throughput protein screening. Noteworthily, the system successfully maintains translation by S-30 cell-free extract below 30 OD. In conclusion, this improved firm cell-free system reduces cost and enables robotic automation and high-throughput thermodynamic analysis, especially for proteins that are difficult to be expressed.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Bioscience and Bioengineering - Volume 112, Issue 2, August 2011, Pages 170–177
نویسندگان
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