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Firm wheat-germ cell-free system with extended vector usage for high-throughput protein screening

Paper ID Volume ID Publish Year Pages File Format Full-Text
21020 43202 2011 8 PDF Available
Title
Firm wheat-germ cell-free system with extended vector usage for high-throughput protein screening
Abstract

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.

Keywords
Polymerase chain reaction; Split-primer; In vitro protein expression; Protein screening; Wheat-germ cell free-system
First Page Preview
Firm wheat-germ cell-free system with extended vector usage for high-throughput protein screening
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 112, Issue 2, August 2011, Pages 170–177
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering