Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems
Date
2015
Authors
Gagoski, D.
Mureev, S.
Giles, N.
Johnston, W.
Dahmer-Heath, M.
Škalamera, D.
Gonda, T.J.
Alexandrov, K.
Editors
Advisors
Journal Title
Journal ISSN
Volume Title
Type:
Journal article
Citation
Journal of Biotechnology, 2015; 195:1-7
Statement of Responsibility
Dejan Gagoski, Sergey Mureev, Nichole Giles, Wayne Johnston, Mareike Dahmer-Heath, Dubravka Škalamera, Thomas J. Gonda, Kirill Alexandrov
Conference Name
Abstract
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.
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Dissertation Note
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Description
Data source: Supplementary data, https://doi.org/10.1016/j.jbiotec.2014.12.006
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© 2014 Elsevier B.V. All rights reserved.