Wheat Cell-free System Research Articles

CF-PA2Vtech: a cell-free human protein array technology for antibody validation against human proteins

Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA2Vtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein.

Autophosphorylation Assays Using Plant Receptor Kinases Synthesized in Cell-Free Systems.

The wheat germ cell-free protein synthesis system has a significant advantage for high-throughput production of eukaryotic multidomain proteins in a folded state. In this chapter, we describe two kinds of methods for performing autophosphorylation assay of plant receptor kinases (PRKs) by using the wheat cell-free system. One is an in vitro kinase assay performed using biotin-streptavidin affinity purification technology, and the other is a luminescence-based high-throughput assay for autophosphorylation analysis. We anticipate that our cell-free-based methods might facilitate the characterization of plant PRKs.

Cell-Free Synthesis of Plant Receptor Kinases.

The wheat germ cell-free protein synthesis system has been used as a eukaryotic protein production system since it was first reported in 1964. Although initially the productivity of this system was not very high, it has now become one of the most versatile protein production systems, thanks to the enhancements made by several groups. In this chapter, we report a protein production method for plant receptor kinases using the wheat cell-free system. We describe a method for the preparation of a cell-free extract from wheat germ, the split-primer PCR method for preparation of transcription templates, and the bilayer cell-free protein synthesis method.

Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues

Protein phosphorylation on Tyr residues is a key post-translational modification in mammals. In plants, recent studies have identified Tyr-specific protein phosphatase and Tyr-phosphorylated proteins in Arabidopsis by phosphoproteomic screenings, implying that plants have a Tyr phosphorylation signal pathway. However, little is known about the protein kinases (PKs) involved in Tyr phosphorylation in plants. Here, we demonstrate that Arabidopsis calcium-dependent protein kinase (CDPK/CPK)-related PKs (CRKs) have high Tyr-autophosphorylation activity and that they can phosphorylate Tyr residue(s) on substrate proteins in Arabidopsis. To identify PKs for Tyr phosphorylation, we examined the autophosphorylation activity of 759 PKs using an Arabidopsis protein array based on a wheat cell-free system. In total, we identified 38 PKs with Tyr-autophosphorylation activity. The CRK family was a major protein family identified. A cell-free substrate screening revealed that these CRKs phosphorylate β-tubulin (TBB) 2, TBB7, and certain transcription factors (TFs) such as ethylene response factor 13 (ERF13). All five CRKs tested showed Tyr-auto/trans-phosphorylation activity and especially two CRKs, CRK2 and CRK3, showed a high ERF13 Tyr-phosphorylation activity. A cell-based transient expression assay revealed that Tyr(16/)Tyr(207) sites in ERF13 were phosphorylated by CRK3 and that Tyr phosphorylation of endogenous TBBs occurs in CRK2 overexpressing cells. Furthermore, crk2 and crk3 mutants showed a decrease in the Tyr phosphorylation level of TBBs. These results suggest that CRKs have Tyr kinase activity, and these might be one of the major PKs responsible for protein Tyr phosphorylation in Arabidopsis plants.

Genome-wide biochemical analysis of Arabidopsis protein phosphatase using a wheat cell-free system

Plant genome possesses over 100 protein phosphatase (PPase) genes that are key regulators of signal transduction via phosphorylation/dephosphorylation event. Here we report a comprehensive functional analysis of protein serine/threonine, dual-specificity and tyrosine phosphatases using recombinant PPases produced by wheat cell-free protein synthesis system. Eighty-two recombinant PPases were successfully produced using Arabidopsis full-length cDNA as templates. In vitro PPase assay was performed using phosphorylated myelin basic protein as substrate. Among the AtPPases examined, 26 serine/threonine, three dual-specificity and one tyrosine PPases exhibited catalytic activity, including 20 serine/threonine and one dual-specificity PPases that showed in vitro activities for the first time. Our study demonstrates genome-wide biochemical analysis of AtPPases using wheat cell-free system, and provides new information and insights on enzyme activities. Structured summary of protein interactionsPTP1dephosphorylatesMBP by phosphatase assay (View interaction).AtPP2CdephosphorylatesMBP by phosphatase assay (View interaction).POLTEdephosphorylatesMBP by phosphatase assay (View interaction).TOPP8dephosphorylatesMBP by phosphatase assay (View interaction).HAB1dephosphorylatesMBP by phosphatase assay (View interaction).ABI2dephosphorylatesMBP by phosphatase assay (View interaction).At1g34750dephosphorylatesMBP by phosphatase assay (View interaction).At1g43900dephosphorylatesMBP by phosphatase assay (View interaction).At3g15260dephosphorylatesMBP by phosphatase assay (View interaction).At5g53140dephosphorylatesMBP by phosphatase assay (View interaction).At1g18030dephosphorylatesMBP by phosphatase assay (View interaction).At3g06270dephosphorylatesMBP by phosphatase assay (View interaction).At2g25070dephosphorylatesMBP by phosphatase assay (View interaction).At3g02750dephosphorylatesMBP by phosphatase assay (View interaction).At5g10740dephosphorylatesMBP by phosphatase assay (View interaction).at4g26080dephosphorylatesMBP by phosphatase assay (View interaction).At4g28400dephosphorylatesMBP by phosphatase assay (View interaction).At5g06750dephosphorylatesMBP by phosphatase assay (View interaction).At4g31860dephosphorylatesMBP by phosphatase assay (View interaction).At3g17250dephosphorylatesMBP by phosphatase assay (View interaction).At4g38520dephosphorylatesMBP by phosphatase assay (View interaction).At3g05640dephosphorylatesMBP by phosphatase assay (View interaction).At5g66080dephosphorylatesMBP by phosphatase assay (View interaction).At1g79630dephosphorylatesMBP by phosphatase assay (View interaction).At2g30170dephosphorylatesMBP by phosphatase assay (View interaction).At5g24940dephosphorylatesMBP by phosphatase assay (View interaction).

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system

BackgroundRecently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.ResultsAtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE.ConclusionFormation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.

Construction of a Protein Library of Arabidopsis Transcription Factors Using a Wheat Cell-Free Protein Production System and Its Application for DNA Binding Analysis

We created a protein library consisting of 647 Arabidopsis transcription factors (TFs) using a wheat cell-free system. The quality of proteins in the library was checked by binding assay of bZIP family proteins. Screening of TFs binding to 5'-regulatory regions of FLC and LFY was conducted using the library, and MYB67 and GBF1 were found to be binding factors.

A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis

BackgroundUbiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.ResultsUsing wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG-tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity.ConclusionIn this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.

The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating calcium signaling. Here, we demonstrate a method for screening substrates phosphorylated by human CaMKIIδ using a wheat cell-free system. The cell-free mixture expressing CaMKIIδ was incubated with HeLa extracts and radiolabeled ATP. From analysis of two-dimensional electrophoresis gels and mass spectrometry, two proteins were found. The cell-free based in vitro kinase assay revealed that CaMKIIδ phosphorylates eukaryotic translation initiation factor 4B and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKIIδ phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKIIδ may regulate subcellular localization of STIP1. This approach may be a useful tool for target screening of protein kinases. Structured summary MINT- 6538664: CAMK2D (uniprotkb: Q13557) phosphorylates (MI: 0217) STIP1 (uniprotkb: P31948) by protein kinase assay (MI: 0424) MINT- 6538652: CAMK2D (uniprotkb: Q13557) phosphorylates (MI: 0217) EIF4B (uniprotkb: P23588) by protein kinase assay (MI: 0424)

Baciphelacin: a new eukaryotic translation inhibitor

Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10 −7 M inhibited protein synthesis by 50% in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin.

Biosynthesis of reovirus-specified polypeptides: Effect of methylation on the efficiency of reovirus genome expression in Vitro

The effect of methylation on the transcription of reovirus genome RNA and the translation of reovirus messenger RNA in vitro was investigated. The following results were obtained: (1) Purified reovirions converted to cores by chymotrypsin treatment catalyzed the transcription of reovirus RNA at comparable rates in the presence of either S-adenosyl- l-methionine (SAdoMet) or S-adenosyl- l-homocysteine (SAdoHcy). Methylated virus reovirus RNA synthesized in the presence of SAdoMet possessed an electrophoretic pattern on agarose-urea-polyacrylamide gels indistinguishable from unmethylated mRNA synthesized in the presence of SAdoHcy. (2) The relative level of SAdoMet-mediated methylation of the four s adn three m class reovirus message species was comparable for methylated mRNA; SAdoHcy at an SAdoHcy:SAdoMet ratio of 350:1 reduced the methylation by more than 98%. (3) Methylated reovirus mRNA was translated by the wheat cell-free system in the presence of either SAdoMet or SAdoHcy with comparable efficiency. Unmethylated mRNA was translated about 50% as efficiently as methylated mRNA in the presence of SAdoMet; in the presence of SAdoHcy, unmethylated mRNA. (4) Polyacrylamide-gel electrophoresis revealed that the σ, μ, and ψ reovirus-specified polypeptides synthesized in vitro in responce to unmethylated mRNA were qualitatively indistiniguishable from those synthesized in response to methylated mRNA, although quantitatively the amount of reovirus polypeptides synthesized with methylated mRNA was much greater. (5) Chemical removal of the 5′-terminal 7-methylguanosine residue of methylated reovirus mRNA by β-elimination reduced the translational activity of the methylated mRNA by about 75%; the translational activity of unmethylated mRNA was reduced only slightly by treatment under identical conditions. (6) Unmethylated reovirus mRNA did not competitively inhibit the translation of methylated mRNA but, rather, appeared to have a cooperative effect. (7) The translation of methylated reovirus mRNA was sensitive to inhibition by 7-methylguanosine 5′-monophosphate (7mGMP), whereas the translation of unmethylated reovirus mRNA was insensitive to 7mGMP; the translational activities of comparable concentrations of methylated and unmethylated mRNA were nearly identical in the presence of 1 m M or greater 7mGMP. (8) The binding of unmethylated reovirus mRNA to wheat ribosomes was detectable by sucrose density centrifugation; however, the binding of methylated mRNA was significantly more efficient than unmethylated mRNA. These results indicate that methylation has no significant effect on the transcription of reovirus RNA in vitro but that the efficiency of translation of reovirus mRNA catalyzed by the wheat system is greatly increased by methylation of the viral mRNA although methylation does not appear to be an obligatory requirement for translation.