title: Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.
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.
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Australia.
authors.forename: Dejan
authors.initials: D
authors.lastname: Gagoski
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Australia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Mureev
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Australia.
authors.forename: Nichole
authors.initials: N
authors.lastname: Giles
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Australia.
authors.forename: Wayne
authors.initials: W
authors.lastname: Johnston
authors.affiliation: University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia.
authors.forename: Mareike
authors.initials: M
authors.lastname: Dahmer-Heath
authors.affiliation: University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia.
authors.forename: Dubravka
authors.initials: D
authors.lastname: Škalamera
authors.affiliation: School of Pharmacy, University of Queensland, Brisbane, Australia; University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gonda
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Australia. Electronic address: k.alexandrov@uq.edu.au.
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007891:Leishmania / Q000166:cytology / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins
keywords: Cell-free protein expression; Gateway cloning; Rolling Circle DNA Amplification; Species Independent Translation Initiation Sequence