Construction of an efficient expression vector for coupled transcription/translation in a wheat germ cell-free system

Sawasaki, Tatsuya; Hasegawa, Yoshinori; Tsuchimochi, Masateru; Kasahara, Yuko; Endo, Yaeta

doi:10.1093/nass/44.1.9

Cited by 21 publications

(22 citation statements)

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“…In this report, we demonstrate that sequences derived from the 5′-UTR (8)(9)(10)(11)(12)(13)(14) and 3′-UTR (25,26) of viral mRNAs, as well as from the abundantly expressed lobster tropomyosin gene (35), are able to function in Drosophila to enhance protein production. By using 5′-and 3′-UTR elements in combination, increases of >20-fold have been achieved, allowing single transgenes to achieve protein expression levels that previously required multiple transgenes, thereby greatly facilitating genetic strain construction.…”

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confidence: 78%

“…In addition, many plant and insect viral mRNAs contain sequences of 20-70 bp in their 5′-UTRs that act in cis to increase translational efficiency (8)(9)(10), one of the most studied being omega (Ω) from tobacco mosaic virus (TMV) (11,12). These translational enhancers can be transferred to other mRNAs and have been widely used to increase protein yields in in vitro extracts derived from cultured insect cells, wheat germ, and rabbit reticulocytes (9,13,14).…”

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confidence: 99%

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Using translational enhancers to increase transgene expression in Drosophila

Pfeiffer

Truman²,

Rubin³

2012

Proc. Natl. Acad. Sci. U.S.A.

385

318

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The ability to specify the expression levels of exogenous genes inserted in the genomes of transgenic animals is critical for the success of a wide variety of experimental manipulations. Protein production can be regulated at the level of transcription, mRNA transport, mRNA half-life, or translation efficiency. In this report, we show that several well-characterized sequence elements derived from plant and insect viruses are able to function in Drosophila to increase the apparent translational efficiency of mRNAs by as much as 20-fold. These increases render expression levels sufficient for genetic constructs previously requiring multiple copies to be effective in single copy, including constructs expressing the temperature-sensitive inactivator of neuronal function Shibire ts1 , and for the use of cytoplasmic GFP to image the fine processes of neurons.

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confidence: 78%

mentioning

confidence: 99%

Using translational enhancers to increase transgene expression in Drosophila

Pfeiffer

Truman²,

Rubin³

2012

Proc. Natl. Acad. Sci. U.S.A.

385

318

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“…In Vitro Expression Constructs-Complete SLS open reading frames (ORFs) were amplified by PCR using specific primers containing 5Ј SgfI and 3Ј PmeI sites for cloning into the 5Ј SgfI and 3Ј EcoICRI sites of the pEU-C-His FlexiVector plasmid (19,20). Pfu high fidelity DNA polymerase (Agilent Technologies, Stratagene, San Diego, CA) was used for all cloning reactions.…”

Section: Methodsmentioning

confidence: 99%

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa

Sevova

Goren²,

Schwartz

et al. 2010

Journal of Biological Chemistry

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The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser 252 ; TbSLS3, Phe 252 ) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems. Sphingolipids (SLs)5 are ubiquitous in eukaryotic membranes, where they perform multiple functions. Besides acting as structural components of biological membranes, they participate in protein sorting and cell signaling via membrane rafts (1) and serve as critical apoptotic and anti-apoptotic second messengers. As such, sphingolipids have important influence in human disease (2). SLs also act as precursors of other essential lipid biosynthetic pathways, e.g. generation of ethanolamine phosphate for use in the Kennedy pathway (3) and subsequent synthesis of phospholipids and glycosylphosphatidyl inositols. The common SL synthesis pathway (Fig. 1) begins with the condensation of serine and palmitate by serine palmitoyl transferase to make the sphingoid base 3-ketodihydrosphinganine, which is then reduced to make sphinganine. Sphinganine is the acceptor for N-fatty acylation by ceramide synthase to make dihydroceramide, which is then desaturated to make ceramide. Ceramide synthase also utilizes sphingosine generated by ceramide turnover. Specific sphingolipid synthases (SLS) then transfer the polar head groups fr...

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“…The AtRGS1 open reading frame and MSP1D1 were cloned into the cell-free pEU expression vector [27] and confirmed by DNA sequencing. Prior to the in vitro transcription reactions, plasmid DNA was subjected to proteinase K treatment as described by Makino et al [28].…”

Section: Methodsmentioning

confidence: 99%

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein

Makino

Beebe

et al. 2016

Protein Expression and Purification

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Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full length RGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Such an understanding can be deduced from in vitro studies using the purified and soluble RGS1-box domain. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.

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Construction of an efficient expression vector for coupled transcription/translation in a wheat germ cell-free system

Cited by 21 publications

References 0 publications

Using translational enhancers to increase transgene expression in Drosophila

Using translational enhancers to increase transgene expression in Drosophila

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein

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