Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system
© Buntru et al.; licensee BioMed Central Ltd. 2014
Received: 7 February 2014
Accepted: 24 April 2014
Published: 3 May 2014
Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase.
The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4–5 h, compared to 4–5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5’ and 3’ untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system.
Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.
KeywordsCell-free protein synthesis In vitro translation Nicotiana tabacum cv. BY-2 Wheat germ extract Protein expression
Cell-free protein synthesis (CFPS) systems based on crude lysates provide several advantages over in vivo systems and offer broad applications in protein engineering, biopharmaceutical product development and post-genomic research . Crude lysates contain the necessary components for translation, protein folding, and energy metabolism, so providing them with amino acids, energy substrates, nucleotides and salts allows almost any protein encoded by a RNA template to be synthesized. In coupled transcription/translation systems supplemented additionally with an appropriate RNA polymerase DNA templates can also be used. In contrast to traditional cell-based expression methods, CFPS offers shorter process times, limited protein hydrolysis and the ability to express toxic proteins or proteins containing specific chemical groups or unnatural amino acids at defined positions . Furthermore, the open nature of the system allows the reaction to be controlled and monitored directly. Although chemical synthesis allows the rapid and controlled synthesis of peptides < 40 residues in length, this is not an economically feasible method for the production of larger proteins .
The most widely used cell-free systems are based on Escherichia coli extract (ECE), wheat germ extract (WGE), rabbit reticulocytes lysate (RLL) and insect cell extract (ICE). These contain diverse cellular components and co-factors that enhance protein expression, folding and modification in different ways. Therefore, the most appropriate system will depend on the origin and the biochemical nature of the target protein. The preparation of ECE is simple and inexpensive, and generally achieves the highest protein yields, from hundreds of micrograms to milligrams per milliliter in batch reactions depending on the target protein [4, 5]. In contrast, eukaryotic systems are less productive and extract preparation is more laborious, but complex proteins can be produced more efficiently and extended post-translational modifications are supported. WGE normally yields tens of micrograms to milligrams of recombinant protein per milliliter, depending on the protein and reaction format [6–8], but extract preparation takes 4–5 d, and the yield of extract from wheat seeds is low . The yields of RLL systems are typically two orders of magnitude lower than WGE  and ICEs prepared from Spodoptera frugiperda can achieve yields of up to 50 μg/mL . Recently two further eukaryotic systems based on CHO cells  and Saccharomyces cerevisae have been described. The CHO extract yield up to 50 μg/mL active firefly luciferase, but the fermentation medium is quit expensive. In contrast the preparation of the yeast extract is inexpensive, but the system produces only low target protein levels of 8 μg/mL active firefly luciferase. The drawbacks of current cell-free systems have therefore created a demand for highly-productive eukaryotic cell-free systems that can be prepared quickly in large amounts.
Here we describe a highly-productive in vitro translation system derived from tobacco BY-2 cells. This type of cells was used due to the eukaryotic nature, the simple and cost-effective fermentation and the well-established genetic modification tools. The novel system was tested against commercial WGEs for the production of three model proteins: enhanced yellow fluorescent protein (eYFP), firefly luciferase (FFLuc) and Renilla reniformis luciferase (RRLuc). By measuring the activity of these optical reporter proteins, the rate of translation was determined without radioactive substrates. Several different 5’ and 3’ untranslated regions were also compared for their translation enhancing activity.
Results and discussion
Preparation of the tobacco Bright Yellow-2 lysate (BYL)
The evacuolation step is comparable to the endosperm removal step during WGE preparation, and is essential because the endosperm contains several translation inhibitors such as tritin and thionin, as well as nucleases and proteases [6, 9, 19]. However, the evacuolation step takes ~90 min, whereas endosperm removal requires 2–3 d because embryo particles remaining after the grinding and sieving of wheat seeds must be selected manually to eliminate those containing larger amounts of endosperm . Without the preselection of embryo particles by flotation using toxic organic solvents such as carbon tetrachloride, the selection process would take even longer .
In vitro translation activity of BYL in batch reactions
The performance of BYL compared to commercial WGEs was investigated by producing the reporter proteins eYFP and luciferase in both systems and measuring fluorescence, which is much faster than standard methods and does not involve radioactive labeling. Kahn et al. and Yukawa et al. demonstrated that the fluorescence of GFP produced in cell-free systems was comparable with the radioactivity of 35S methionine incorporated during the translation process, showing that translation yield can be quantified by fluorescence measurement. Similarly, a correlation between luciferase activity and the incorporation of 14C leucine has been demonstrated in WGE, RLL and ECE [23, 24].
In vitro translation activity of BYL in dialysis reactions
In summary, the BYL is a promising system with high translational activity, short preparation time and high potential for scaling-up. It is likely that the efficiency of the BYL system can be improved further, e.g. by optimizing reaction conditions through a factorial design approach . The BYL system is based on eukaryotic components and should therefore be suitable for the expression of eukaryotic proteins, but further studies are required to determine whether the BYL system is capable of carrying out modifications such as glycosylation and the formation of disulfide bonds. A coupled transcription/translation system based on the BYL shall also be addressed in further studies.
Tobacco cells (Nicotiana tabacum L. cv. Bright Yellow 2, BY-2) were cultivated continuously in a 5-L fermenter (Type 100e, Applicon Biotechnology, AC Schiedam, Netherlands) with a 20% packed cell volume at 26°C in the dark in Murashige-Skoog liquid medium (Murashige and Skoog Basal Salt Mixture, Duchefa Biochemie, Haarlem, Netherlands) supplemented with 3% (w/v) sucrose, 1 mg/L thiamine-HCl, 0.2 mg/L 2,4 dichlorophenoxyacetic acid and 100 mg/L myo-inositol.
Preparation of the BY-2 cell lysate
The preparation of lysate from evacuolated BY-2 protoplasts was carried out as described by Komoda et al. and Gursinsky et al. with significant modifications. Protoplasts were prepared from cells in the exponential growth phase of a continuous fermentation at a constant packed cell volume of 20% by treating the cells with 3% (v/v) Rohament CL, 2% (v/v) Rohament PL, and 0.1% (v/v) Rohapect UF (all from AB Enzymes, Darmstadt, Germany) in 0.37 mM mannitol, 5 mM CaCl2, 12.5 mM sodium acetate (pH 5.8) for 1.5 h. The resulting protoplasts were layered onto a discontinuous Percoll gradient containing (from bottom to top) 70% (v/v, 3 ml), 40% (v/v, 5 ml), 30% (v/v, 3 ml), 15% (v/v, 3 ml) and 0% (3 ml) Percoll (GE Healthcare, Munich, Germany) in 0.7 M mannitol, 20 mM MgCl2, and 5 mM PIPES-KOH (pH 7.0). After centrifugation at 12,000 g for 1 h at 25°C in a swinging bucket rotor, evacuolated protoplasts were recovered from the 40-70% (v/v) Percoll solution interface. The evacuolated protoplasts were suspended in three volumes of TR buffer (30 mM HEPES-KOH (pH 7.4), 80 mM potassium acetate, 0.5 mM magnesium acetate, 2 mM DTT) supplemented with one tablet per 50 ml of Complete EDTA-free Protease Inhibitor Mixture (Roche Diagnostics, Mannheim, Germany) and disrupted using the nitrogen decompression method in a cell disruption vessel (Parr Instrument, Frankfurt, Germany) for 30 min at 10 bar. Nuclei and non-disrupted cells were removed by centrifugation at 500 g for 10 min at 4°C. The supernatant was supplemented with 0.5 mM CaCl2 and treated with 75 U/ml nuclease S7 (Roche Diagnostics) for 15 min at 20°C. The lysate was supplemented with 2 mM EGTA as chelating agent for the Ca2+ ions to inactivate the nuclease and frozen at -80°C in 1-ml aliquots.
DNA template preparation
Plasmid pIVEX1.3_eYFP-His was kindly provided by Dr. Stefan Kubick (Fraunhofer Institute for Biomedical Engineering, Potsdam-Golm, Germany). Plasmid pIVEX_GAA_Omega_eYFP-His was prepared by inserting annealed oligonucleotides 1 and 2 (Additional file 2) containing the T7 promoter and the TMV 5’ leader sequence (omega) into pIVEX1.3_eYFP-His using the NspI and NcoI sites. Plasmid pIVEX_GAA_E02_eYFP-His was prepared by inserting annealed oligonucleotides 3 and 4 (Additional file 2) containing the T7 promoter and the synthetic 5’ UTR E02 into pIVEX1.3_eYFP-His using the NspI and NcoI sites. Plasmids pIX4.0_His-eYFP, pIVEX1.3_His-eYFP, pF3A_His-eYFP and pCITE2a_His-eYFP were generated by amplifying the His-eYFP sequence from pIX3.0_His-eYFP (kindly provided by Dr. Stefan Kubick) using the following primers (Additional file 2): 5 and 6 for cloning into pIX4.0_eCFP (kindly provided by Dr. Stefan Kubick), 5 and 7 for cloning into pIVEX1.3_eYFP-His and pF3A (Promega, Mannheim, Germany), and 5 and 8 for cloning into pCITELuc (kindly provided by Dr. Sven-Erik Behrens, Institute of Biochemistry and Biotechnology, Halle/Saale, Germany). The PCR products were digested with BspHI and NotI, BspHI and KpnI, and BspHI and XbaI, respectively, and introduced into the NcoI and NotI sites of pIX4.0_eCFP, the NcoI and KpnI sites of pIVEX1.3_eYFP-His and pF3A, and the NcoI and XbaI sites of pCITELuc. Omega_eYFP-His was generated using a two-step PCR procedure (Sawasaki et al., 2007). In the first step, eYFP-His was amplified using pIVEX1.3_eYFP-His as a template and the two gene-specific primers 9 and 10 (Additional file 2). In the second step, the T7 promoter and omega sequence were fused to eYFP-His using primers 11 (5’ end of T7 promoter), 12 (3’ end of T7 promoter and omega sequence) and 10 as the previous step. For vector pIVEX1.3_FFLuc-His, the FFLuc gene was amplified by PCR using pCITELuc as the template and primers 17 and 18 (Additional file 2). The product was digested with NcoI and XhoI and reintroduced into pCITELuc in-frame with the His-tag to generate pCITE2a_FFLuc-His. Then the FFLuc-His gene was amplified using primers 17 and 19 and introduced into the NcoI and KpnI sites of pIVEX1.3_eYFP-His. The RRLuc gene was amplified by PCR using pSP_RRLuc (kindly provided by Dr. Sven-Erik Behrens) as the template and primers 20 and 21 (Additional file 2). The product was digested with NcoI and XhoI and inserted into pCITELuc to generate pCITE2a_RRLuc-His. The RRLuc-His sequence was then amplified using primers 20 and 21 and inserted into the NcoI and KpnI sites of pIVEX1.3_eYFP-His to generate pIVEX1.3_RRLuc-His. Plasmids pIVEX1.3_eYFP-His_Poly(A) and pIVEX_GAA_Omega_eYFP-His_Poly(A) were generated by amplifying the 3’-UTR from pIX4.0_His-eYFP by PCR using primers 22 and 23 and inserting the product into the KpnI and EcoRI sites of pIVEX1.3_eYFP-His and pIVEX_GAA_Omega_eYFP-His.
In vitro transcription
Capped mRNA was transcribed in vitro in the presence of the cap analogs m7G[5’]ppp[5’]G or m27,3’-OG[5’]ppp[5’]G (New England Biolabs, Ipswich, MA, USA) using the T7 High Yield RNA Synthesis Kit (New England Biolabs) and DNA templates amplified by PCR from the constructs described above using Phusion Polymerase (New England Biolabs) and the following primers (Additional file 2): 10 and 13 for pIVEX1.3_eYFP-His, pIVEX1.3_His-eYFP, pCITE2a_His-eYFP, pIVEX1.3_FFLuc-His and pIVEX1.3_RRLuc-His; 10 and 14 for pIVEX_GAA_Omega_eYFP-His, pIVEX_GAA_E02_eYFP-His and pIX4.0_His-eYFP; 10 and 11 for Omega_eYFP-His, and 15 and 16 for pF3A_His-eYFP. The RNA was purified using the DyeEx 2.0 Spin Kit (Qiagen, Hilden, Germany).
In vitro translation
Batch reactions contained 50% (v/v) BYL, 30% (v/v) modified TR buffer (30 mM HEPES-KOH, pH 7.6, 80 mM potassium acetate, 2 mM DTT), 0.75 mM ATP, 0.1 mM GTP, 25 mM creatine phosphate, 50 μM of each amino acid, 80 μM spermine and 0.2 mg/mL creatine phosphokinase (Roche Diagnostics). The magnesium concentration was adjusted with magnesium acetate to 1.44 mM and the potassium concentration was adjusted with potassium acetate to 61 mM or 68 mM, according to the expression construct. Any magnesium and potassium already present in the BY-2 cell extracts was ignored. The batch translation reactions were carried out at 25°C and 500 rpm for 18 h in a thermomixer (Eppendorf, Hamburg, Germany). WGE (Promega) translation reactions were carried out according to the manufacturer’s instructions.
Dialysis reactions were carried out in RTS 100 Wheat Germ continuous exchange cell-free (CECF) devices (5Prime, Hamburg, Germany) using the same mixtures described for the batch reactions except that we also added 40 units of murine RNase Inhibitor (New England Biolabs). Feeding solutions contained 67.5% (v/v) modified TR buffer. For the BYL system this was 30 mM HEPES-KOH (pH 7.6), 90 mM potassium acetate, 2.1 mM magnesium acetate and 2 mM DTT, supplemented with 0.75 mM ATP, 0.1 mM GTP, 25 mM creatine phosphate, 50 μM of each amino acid and 80 μM spermine. For the Promega WGE system this was 30 mM HEPES-KOH (pH 7.6), 200 mM potassium acetate, 3.1 mM magnesium acetate and 7.4 mM DTT, supplemented with 1.2 mM ATP, 0.1 mM GTP, 10 mM creatine phosphate, 80 μM of each amino acid and 500 μM spermidine. Translation reactions in dialysis mode were carried out at 25°C and 900 rpm for 18 h in a thermomixer (Eppendorf). Translation reactions with the RTS 100 Wheat Germ CECF Kit (5Prime) were carried out according to the manufacturer’s instructions.
The fluorescence signal from eYFP was quantified using a Synergy HT Multi-Mode Microplate Reader (Biotek, Bad Friedrichshall, Germany) with 485/20 nm excitation and 528/20 nm emission filters. The quantity of eYFP was determined by generating a standard curve based on different concentrations of eYFP in BYL translation reactions without a mRNA template. The eYFP standard was produced in a home-made E. coli in vitro translation system and purified by immobilized metal-affinity chromatography (IMAC) and size exclusion chromatography (SEC). The concentration of protein was determined using a colorimetric assay.
Firefly and Renilla luciferase activities were measured with the Luciferase Assay System (Promega) and a GENios Pro microplate reader (Tecan, Mainz-Kastel, Germany). Firefly luciferase purchased from Roche Diagnostics was used as a standard.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was performed using precast NuPAGE 4-12% polyacrylamide Bis-Tris gels (Life Technologies, Carlsbad, CA, USA). The gel was stained with Coomassie brilliant blue R-250. PageRuler Prestained Protein Ladder (Thermo Scientific, Waltham, MA, USA) was used as molecular weight marker.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional file.
Barley yellow dwarf virus
Continuous exchange cell-free
Cell-free protein synthesis
Enhanced yellow fluorescent protein
Insect cell extract
Immobilized metal-affinity chromatography
Rabbit reticulocyte lysate
Renilla reniformis luciferase
Size exclusion chromatography
Tobacco mosaic virus
Wheat germ extract.
We would like to thank Dr. Stefan Kubick (Fraunhofer Institute for Biomedical Engineering, Potsdam-Golm, Germany) for providing the vectors pIVEX1.3_eYFP-His, pIX4.0_eCFP and pIX3.0_His-eYFP. We also thank Dr. Sven-Erik Behrens (Institute of Biochemistry and Biotechnology, Halle/Saale, Germany) for providing the plasmids pCITELuc and pSP_RRLuc. Furthermore we would like to thank Dr. Tanja Holland (Fraunhofer Institute for Molecular Biology and Applied Ecology IME/IPP, Aachen, Germany) for her help with the BY-2 cell cultures. This work was funded by the Federal Ministry of Education and Research (BMBF, FKZ 0315942) and the Fraunhofer Society.
- Swartz JR: Transforming biochemical engineering with cell-free biology. AIChE Journal. 2012, 58: 5-13. 10.1002/aic.13701.View ArticleGoogle Scholar
- White ER, Reed TM, Ma Z, Hartman MCT: Replacing amino acids in translation: Expanding chemical diversity with non-natural variants. Methods. 2013, 60: 70-74. 10.1016/j.ymeth.2012.03.015.View ArticleGoogle Scholar
- Nilsson BL, Soellner MB, Raines RT: Chemical synthesis of proteins. Annu Rev Biophys Biomol Struct. 2005, 34: 91-118. 10.1146/annurev.biophys.34.040204.144700.View ArticleGoogle Scholar
- Kim HC, Kim TW, Kim DM: Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source. Process Biochem. 2011, 46 (6): 1366-1369. 10.1016/j.procbio.2011.03.008.View ArticleGoogle Scholar
- Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, Gold DS, Heinsohn HG, Murray CJ: Microscale to manufacturing scale-up of cell-free cytokine production–a new approach for shortening protein production development timelines. Biotechnol Bioeng. 2011, 108 (7): 1570-1578. 10.1002/bit.23103.View ArticleGoogle Scholar
- Madin K, Sawasaki T, Ogasawara T, Endo Y: A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc Natl Acad Sci USA. 2000, 97 (2): 559-564. 10.1073/pnas.97.2.559.View ArticleGoogle Scholar
- Slater MR, Hurst R, Pferdehirt B, White D, Niles A, Betz N, Schenborn E: Expression of Soluble Native Human Proteins in Cell-Free Extracts. Promega Notes. 2005, 91: 22-25.Google Scholar
- Zhao KQ, Hurst R, Slater MR, Bulleit RF: Functional protein expression from a DNA based wheat germ cell-free system. J Struct Funct Genomics. 2007, 8 (4): 199-208. 10.1007/s10969-007-9035-2.View ArticleGoogle Scholar
- Takai K, Sawasaki T, Endo Y: Practical cell-free protein synthesis system using purified wheat embryos. Nature protocols. 2010, 5 (2): 227-238. 10.1038/nprot.2009.207.View ArticleGoogle Scholar
- Jackson M, Boutell J, Cooley N, He M: Cell-free protein synthesis for proteomics. Briefings in functional genomics and proteomics. 2004, 2: 308-319. 10.1093/bfgp/2.4.308.View ArticleGoogle Scholar
- Ezure T, Suzuki T, Shikata M, Ito M, Ando E: A cell-free protein synthesis system from insect cells. Methods Mol Biol. 2010, 607: 31-42. 10.1007/978-1-60327-331-2_4.View ArticleGoogle Scholar
- Brodel AK, Sonnabend A, Kubick S: Cell-free protein expression based on extracts from CHO cells. Biotechnol Bioeng. 2014, 111 (1): 25-36. 10.1002/bit.25013.View ArticleGoogle Scholar
- Hodgman CE, Jewett MC: Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis. Biotechnol Bioeng. 2013, 110 (10): 2643-2654. 10.1002/bit.24942.View ArticleGoogle Scholar
- Carlson ED, Gan R, Hodgman CE, Jewett MC: Cell-free protein synthesis: applications come of age. Biotechnol Adv. 2012, 30 (5): 1185-1194. 10.1016/j.biotechadv.2011.09.016.View ArticleGoogle Scholar
- Komoda K, Naito S, Ishikawa M: Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts. Proc Natl Acad Sci USA. 2004, 101 (7): 1863-1867. 10.1073/pnas.0307131101.View ArticleGoogle Scholar
- Gursinsky T, Schulz B, Behrens SE: Replication of Tomato bushy stunt virus RNA in a plant in vitro system. Virology. 2009, 390 (2): 250-260. 10.1016/j.virol.2009.05.009.View ArticleGoogle Scholar
- Ishibashi K, Komoda K, Ishikawa M: In vitro translation and replication of tobamovirus RNA in a cell-free extract of evacuolated tobacco BY-2 protoplasts. 2006, Berlin: SpringerView ArticleGoogle Scholar
- Loewus MW, Loewus F: The Isolation and Characterization of d-Glucose 6-Phosphate Cycloaldolase (NAD-Dependent) from Acer pseudoplatanus L. Cell Cultures: Its Occurrence in Plants. Plant Physiol. 1971, 48 (3): 255-260. 10.1104/pp.48.3.255.View ArticleGoogle Scholar
- Endo Y, Sawasaki T: High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. Biotechnol Adv. 2003, 21 (8): 695-713. 10.1016/S0734-9750(03)00105-8.View ArticleGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3.View ArticleGoogle Scholar
- Kahn TW, Beachy RN, Falk MM: Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo. Curr Biol. 1997, 7 (4): R207-208. 10.1016/S0960-9822(06)00100-X.View ArticleGoogle Scholar
- Yukawa M, Kuroda H, Sugiura M: A new in vitro translation system for non-radioactive assay from tobacco chloroplasts: effect of pre-mRNA processing on translation in vitro. Plant J. 2007, 49 (2): 367-376. 10.1111/j.1365-313X.2006.02948.x.View ArticleGoogle Scholar
- Kolb VA, Makeyev EV, Spirin AS: Folding of firefly luciferase during translation in a cell-free system. The EMBO journal. 1994, 13 (15): 3631-3637.Google Scholar
- Kolb VA, Makeyev EV, Spirin AS: Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system. J Biol Chem. 2000, 275 (22): 16597-16601. 10.1074/jbc.M002030200.View ArticleGoogle Scholar
- Kozak M: Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene. 2005, 361: 13-37.View ArticleGoogle Scholar
- Fan Q, Treder K, Miller WA: Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency. BMC Biotechnol. 2012, 12: 22-10.1186/1472-6750-12-22.View ArticleGoogle Scholar
- Sonenberg N: mRNA translation: influence of the 5' and 3' untranslated regions. Curr Opin Genet Dev. 1994, 4 (2): 310-315. 10.1016/S0959-437X(05)80059-0.View ArticleGoogle Scholar
- Sachs AB, Sarnow P, Hentze MW: Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell. 1997, 89 (6): 831-838. 10.1016/S0092-8674(00)80268-8.View ArticleGoogle Scholar
- Herrshey JWB, Merrick WC: The pathway and mechanism of initiation of protein synthesis. 2000, New York: Cold Spring HarborGoogle Scholar
- Dreher TW, Miller WA: Translational control in positive strand RNA plant viruses. Virology. 2006, 344 (1): 185-197. 10.1016/j.virol.2005.09.031.View ArticleGoogle Scholar
- Kneller EL, Rakotondrafara AM, Miller WA: Cap-independent translation of plant viral RNAs. Virus Res. 2006, 119 (1): 63-75. 10.1016/j.virusres.2005.10.010.View ArticleGoogle Scholar
- Nicholson BL, White KA: 3' Cap-independent translation enhancers of positive-strand RNA plant viruses. Curr Opin Virol. 2011, 1 (5): 373-380. 10.1016/j.coviro.2011.10.002.View ArticleGoogle Scholar
- Walsh D, Mohr I: Viral subversion of the host protein synthesis machinery. Nat Rev Microbiol. 2011, 9 (12): 860-875. 10.1038/nrmicro2655.View ArticleGoogle Scholar
- Elroy-Stein O, Fuerst TR, Moss B: Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci USA. 1989, 86 (16): 6126-6130. 10.1073/pnas.86.16.6126.View ArticleGoogle Scholar
- Parks GD, Duke GM, Palmenberg AC: Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region. J Virol. 1986, 60 (2): 376-384.Google Scholar
- Wang S, Miller WA: A sequence located 4.5 to 5 kilobases from the 5' end of the barley yellow dwarf virus (PAV) genome strongly stimulates translation of uncapped mRNA. J Biol Chem. 1995, 270 (22): 13446-13452. 10.1074/jbc.270.22.13446.View ArticleGoogle Scholar
- Ding H, Griesel C, Nimtz M, Conradt HS, Weich HA, Jager V: Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system. Protein Expr Purif. 2003, 31 (1): 34-41. 10.1016/S1046-5928(03)00138-4.View ArticleGoogle Scholar
- Suzuki T, Ito M, Ezure T, Kobayashi S, Shikata M, Tanimizu K, Nishimura O: Performance of expression vector, pTD1, in insect cell-free translation system. J Biosci Bioeng. 2006, 102 (1): 69-71. 10.1263/jbb.102.69.View ArticleGoogle Scholar
- Wickham TJ, Davis T, Granados RR, Shuler ML, Wood HA: Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnol Prog. 1992, 8 (5): 391-396. 10.1021/bp00017a003.View ArticleGoogle Scholar
- Yamaji H, Hirakawa D, Tagai S, Fukuda H: Production of protein kinase C-delta by the baculovirus-insect cell system in serum-supplemented and serum-free media. J Biosci Bioeng. 2003, 95 (2): 185-187.View ArticleGoogle Scholar
- Akbergenov RZ, Zhanybekova SS, Kryldakov RV, Zhigailov A, Polimbetova NS, Hohn T, Iskakov BK: ARC-1, a sequence element complementary to an internal 18S rRNA segment, enhances translation efficiency in plants when present in the leader or intercistronic region of mRNAs. Nucleic Acids Res. 2004, 32 (1): 239-247. 10.1093/nar/gkh176.View ArticleGoogle Scholar
- Matveeva OV, Shabalina SA: Intermolecular mRNA-rRNA hybridization and the distribution of potential interaction regions in murine 18S rRNA. Nucleic Acids Res. 1993, 21 (4): 1007-1011. 10.1093/nar/21.4.1007.View ArticleGoogle Scholar
- Sarge KD, Maxwell ES: Evidence for a Competitive-Displacement Model for the initiation of protein synthesis involving the intermolecular hybridization of 5 S rRNA, 18 S rRNA and mRNA. FEBS Lett. 1991, 294 (3): 234-238. 10.1016/0014-5793(91)81437-D.View ArticleGoogle Scholar
- Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TM: The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res. 1987, 15 (8): 3257-3273. 10.1093/nar/15.8.3257.View ArticleGoogle Scholar
- Zaccomer B, Haenni AL, Macaya G: The remarkable variety of plant RNA virus genomes. J Gen Virol. 1995, 76 (Pt 2): 231-247.View ArticleGoogle Scholar
- Gallie DR, Walbot V: Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation. Nucleic Acids Res. 1992, 20 (17): 4631-4638. 10.1093/nar/20.17.4631.View ArticleGoogle Scholar
- Gallie DR: Translational control of cellular and viral mRNAs. Plant Mol Biol. 1996, 32 (1–2): 145-158.View ArticleGoogle Scholar
- Sawasaki T, Ogasawara T, Morishita R, Endo Y: A cell-free protein synthesis system for high-throughput proteomics. Proc Natl Acad Sci USA. 2002, 99 (23): 14652-14657. 10.1073/pnas.232580399.View ArticleGoogle Scholar
- Kamura N, Sawasaki T, Kasahara Y, Takai K, Endo Y: Selection of 5'-untranslated sequences that enhance initiation of translation in a cell-free protein synthesis system from wheat embryos. Bioorg Med Chem Lett. 2005, 15 (24): 5402-5406. 10.1016/j.bmcl.2005.09.013.View ArticleGoogle Scholar
- Kozak M: How strong is the case for regulation of the initiation step of translation by elements at the 3' end of eukaryotic mRNAs?. Gene. 2004, 343 (1): 41-54. 10.1016/j.gene.2004.08.011.View ArticleGoogle Scholar
- Vasilev N, Gromping U, Lipperts A, Raven N, Fischer R, Schillberg S: Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method. Plant Biotechnol J. 2013, 11 (7): 867-874. 10.1111/pbi.12079.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.