Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systemsJournal of Biotechnology


Dejan Gagoski, Sergey Mureev, Nichole Giles, Wayne Johnston, Mareike Dahmer-Heath, Dubravka Škalamera, Thomas J. Gonda, Kirill Alexandrov
Applied Microbiology and Biotechnology / Biotechnology


Recombinant production of a difficult-to-express eukaryotic protein in a bacterial expression system

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Wei Li, Zifang Song, Qichang Zheng, Jun Xiong, Dan Shang, Siming Guan, Xiaogang Shu

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K. Gloux, M. Leclerc, H. Iliozer, R. L'Haridon, C. Manichanh, G. Corthier, R. Nalin, H. M. Blottiere, J. Dore

Fluorescent probe for high-throughput screening of membrane protein expression

A. E. Backmark, N. Olivier, A. Snijder, E. Gordon, N. Dekker, A. D. Ferguson

A High-Throughput-Compatible 3D Microtissue Co-Culture System for Phenotypic RNAi Screening Applications

C. R. Thoma, S. Stroebel, N. Rosch, B. Calpe, W. Krek, J. M. Kelm


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Journal of Biotechnology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Biotechnology j ourna l ho me page: www.elsev ier .com/ locate / jb io tec

Gateway-compatible vectors for high-throughpu in proDejan Ga JohQ1

Mareike Gon a Institute for M b University of c School of Pha a r t i c l

Article history:

Received 13 A

Received in re

Accepted 11 D

Available onlin


Gateway cloning

Cell-free protein expression

Species Independent Translation Initiation


Rolling Circle DNA Amplification otein alyz lonin uite o quenc 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 1. Introdu

The emeQ2 transforme to genomic sequencing analyze th putative op (UniProt, 20

One of recombinan of research inherently u to express gation, mis issues, cell ∗ Correspon

Australia. Tel.:

E-mail add 1 These auth http://dx.doi.o 0168-1656/© 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38e this article in press as: Gagoski, D., et al., Gateway-compatible vectors for high-throughput protein expression in pro- and c cell-free systems. J. Biotechnol. (2014), 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. © 2014 Published by Elsevier B.V. ction rgence of next generation sequencing technologies has d biology by providing rapid and inexpensive access information. However, the ability to generate gene data greatly outstrips our ability to experimentally e encoded polypeptides. Currently less than 5% of en reading frames have been experimentally studied 12). the key hurdles to progress in this direction is the t production of proteins. Despite more than 40 years , recombinant protein expression is a complex and npredictable process. It is often difficult or impossible functional proteins due to their host toxicity, aggrefolding or degradation. In an effort to address these -free protein expression (CFPE) systems have been ding author at: Building 80, 306 Carmody Rd, St. Lucia, 4072 QLD, +61 7 3346 2017. ress: (K. Alexandrov). ors contributed equally to this work. increasingly employed for recombinant protein production. In

CFPE a translation-competent cellular extract is primed with DNA or RNA that encodes the gene of interest (Jermutus et al., 1998;

Whittaker, 2013).

The commonly employed Escherichia coli and wheat germ cellfree systems have been optimized for production of preparative amounts of “hard-to-express” proteins. Concomitantly there has been an expansion in the species used as lysate sources for CFPE.

Common eukaryotic sources are Sf-9 insect cells, mammalian cells, rabbit reticulocytes, and most recently the ciliated protozoan Leishmania tarentolae (Mureev et al., 2009). These systems have been used for expression of proteins with unnatural amino acids, production of protein complexes, construction of protein arrays and even expression of membrane proteins (Albayrak and Swartz, 2013;

Haberstock et al., 2012; He and Taussig, 2008; Kuruma et al., 2010).

Depending on the CFPE configuration, in vitro translation reactions can be primed either with mRNA, or in the case of coupled transcription-translation systems, with the template DNA (Katzen et al., 2005). The coupled CFPE systems are most suitable for multiplexing and high throughput applications due to their single tube/single step format (Sitaraman and Chatterjee, 2009). Such format for instance enables expression of transcriptomes (Goshima rg/10.1016/j.jbiotec.2014.12.006 2014 Published by Elsevier B.V. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 and eukaryotic cell-free systems goskia,1, Sergey Mureeva,1, Nichole Gilesa, Wayne

Dahmer-Heathb, Dubravka Sˇkalamerab, Thomas J. olecular Bioscience, University of Queensland, Australia

Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia rmacy, University of Queensland, Brisbane, Australia e i n f o ugust 2014 vised form 6 December 2014 ecember 2014 e xxx a b s t r a c t

Although numerous techniques for pr sequencing outstrips our ability to an established a system for parallelized c of proteins. This system is based on a s

Independent Translation Initiation Set protein expression nstona, dac,b, Kirill Alexandrova,∗ expression and production are available the pace of genome e the encoded proteins. To address this bottleneck, we have g, DNA production and cell-free expression of large numbers f pCellFree Gateway destination vectors that utilize a Species e (SITS) that mediates recombinant protein expression in any

Please cit ector eukaryoti /j.jbi

ARTICLE IN PRESSG ModelBIOTEC 6956 1–7 2 D. Gagoski et al. / Journal of Biotechnology xxx (2014) xxx–xxx et al., 2008), construction of protein microarrays (Zarate and

Galbraith, 2014) or directed evolution and library selection experiments (Odegrip et al., 2004). However, rapid and inexpensive cloning of large gene collections requires multiplexing and miniaturization o concentrati plate prepa have been d mat than clo problem is tionally uni modules fo developing (SITS) that systems (M