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Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems

Paper ID Volume ID Publish Year Pages File Format Full-Text
22932 43401 2015 7 PDF Available
Title
Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems
Abstract

•A suite of universal Gateway-based in vitro expression vectors was constructed.•The performance of vectors was tested in E. coli and Leishmania cell-free systems.•Rolling Circle DNA Amplification was used to prepare templates for in vitro translation.

Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era.

Keywords
Gateway cloning; Cell-free protein expression; Species Independent Translation Initiation Sequence; Rolling Circle DNA Amplification
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Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 195, 10 February 2015, Pages 1–7
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us