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Готові списки джерел за темами / Proteins Synthesis

Добірка наукової літератури з теми "Proteins Synthesis"

Автор: Grafiati

Опубліковано: 4 червня 2021

Оновлено: 29 січня 2023

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Статті в журналах з теми "Proteins Synthesis":

1

Nilsson, Bradley L., Matthew B. Soellner, and Ronald T. Raines. "Chemical Synthesis of Proteins." Annual Review of Biophysics and Biomolecular Structure 34, no. 1 (June 2005): 91–118. http://dx.doi.org/10.1146/annurev.biophys.34.040204.144700.

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2

Gibney, Brian R., Francesc Rabanal, and P. Leslie Dutton. "Synthesis of novel proteins." Current Opinion in Chemical Biology 1, no. 4 (December 1997): 537–42. http://dx.doi.org/10.1016/s1367-5931(97)80050-6.

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3

Mejuch, Tom, and Herbert Waldmann. "Synthesis of Lipidated Proteins." Bioconjugate Chemistry 27, no. 8 (August 2016): 1771–83. http://dx.doi.org/10.1021/acs.bioconjchem.6b00261.

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4

Borgia, Jeffrey A., and Gregg B. Fields. "Chemical synthesis of proteins." Trends in Biotechnology 18, no. 6 (June 2000): 243–51. http://dx.doi.org/10.1016/s0167-7799(00)01445-1.

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5

Hilvert, Donald. "Chemical synthesis of proteins." Chemistry & Biology 1, no. 4 (December 1994): 201–3. http://dx.doi.org/10.1016/1074-5521(94)90011-6.

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6

Kohen, Amnon, Priyanka Singh, and Qi Guo. "Chemoenzymatic Synthesis of Ubiquitous Biological Redox Cofactors." Synlett 28, no. 10 (April 10, 2017): 1151–59. http://dx.doi.org/10.1055/s-0036-1588768.

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Redox cofactors are utilized by a myriad of proteins, ranging from metabolic enzymes to those performing post-translational modifications. Labeled redox cofactors have served as a vital tool for a broad range of studies. This account describes chemoenzymatic syntheses of the isotopically labeled, biologically important redox cofactors: nicotinamide adenine dinucleotide, methylene tetrahydrofolate, and flavin nucleotides. An overview of the general strategy is presented. These examples demonstrate the utility of enzymatic synthesis.1 Introduction2 Nicotinamide Cofactors2.1 Synthesis of Remote-Labeled 14C-NADPH2.1.1 Synthesis of [Ad-14C]NADPH2.1.2 Synthesis of [Carbonyl-14C]NADPH2.2 Synthesis of S- and R-[4-3H]NADPH2.2.1 One-Step S- and Three-Step R-[4-3H]NADPH Synthesis2.2.2 One-Pot, One-Step R-[4-3H]NADPH Synthesis2.3 Synthesis of S- and R-[Ad-14C, 4-2H]NADPH2.3.1 One-Step S-, Three-Step R-[Ad-14C, 4-2H]NADPH Synthesis2.3.2 One-Pot, One-Step R-[Ad-14C, 4-2H]NADPH Synthesis3 Methylene Tetrahydrofolate4 Flavin Nucleotides5 Conclusions and Outlook

7

Kent, Stephen B. H. "Total chemical synthesis of proteins." Chem. Soc. Rev. 38, no. 2 (2009): 338–51. http://dx.doi.org/10.1039/b700141j.

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8

Tam, James P., and Clarence T. T. Wong. "Chemical Synthesis of Circular Proteins." Journal of Biological Chemistry 287, no. 32 (June 14, 2012): 27020–25. http://dx.doi.org/10.1074/jbc.r111.323568.

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9

Muir, Tom W., and Stephen B. H. Kent. "The chemical synthesis of proteins." Current Opinion in Biotechnology 4, no. 4 (August 1993): 420–27. http://dx.doi.org/10.1016/0958-1669(93)90007-j.

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10

Huang, Yichao, and Lei Liu. "Chemical synthesis of crystalline proteins." Science China Chemistry 58, no. 12 (July 8, 2015): 1779–81. http://dx.doi.org/10.1007/s11426-015-5462-2.

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Дисертації з теми "Proteins Synthesis":

1

Baas, Tracey Lynn. "The design, synthesis, and characterization of template assembled synthetic proteins /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/11561.

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2

Draffan, Lynda Catherine. "Chemical synthesis of proteins." Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/13715.

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3

Morton, Gail Helen. "The chemical synthesis of proteins." Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/15437.

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The viability of extending the present methodology designed for the solid phase synthesis of peptides has been investigated. Using the catalytic domain of stromelysin (SCD. 173 residues) as the model system, a number of different factors affecting the preparation and purification of chemically synthesised proteins are examined. The purification SCD, prepared using stepwise solid phase synthesis is described. Following characterisation, it is evident that protein of the correct primary sequence has been prepared, furthermore, preliminary studies centred on the enzymatic activity of SCD indicate that active protease has been isolated. However, comparison of the conformational and biophysical properties of chemically synthesised SCD with the recombinant counterpart, suggests that there are problems associated with the folding of the synthetic SCD. The construction of chemically synthesised SCD via convergent protein synthesis is also described. Two different coupling strategies involving classical and azide fragment condensation are examined where it has been highlighted that the overall success of each fragment coupling is greatly dependent on the peptide length and sequence. As well as comparing methods for the preparation and coupling of fully and minimally protected peptides, general procedures for both solution and solid phase fragment coupling are discussed. A novel strategy for the convergent synthesis of peptides and proteins has been investigated. In this total chemical synthesis, two minimally protected peptides are joined through unique, mutually reactive functional groups, yielding a peptide analogue with a thioether replacement for the native peptide bond at the site of ligation. A general route to C-terminal sulfhydryl and N-terminal haloacetylated peptides is presented, accompanied with results of the preliminary ligation studies.

4

Walker, Douglas Gordon. "Characterization of immediate-early and early proteins of murine cytomegalovirus synthesized in permissive and nonpermissive cells." Thesis, University of British Columbia, 1985. http://hdl.handle.net/2429/25985.

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The gene products produced by murine cytomegalovirus (MCMV) in infected cells prior to viral DNA synthesis are believed to control the interaction of the virus with the cells, determining whether a permissive infection results, with virus replication, or whether further virus gene expression is inhibited, resulting in a latent or abortive infection. The aim of this study was to characterize the early viral gene products that are produced in permissive and nonpermissive cells. The proteins produced in 3T3-L1 cells, permissively infected with MCMV, during the first six hours of infection (the period prior to viral DNA replication) were characterized by polyacrylamide gel electrophoresis. Ten of the proteins were classified as immediate-early (IE) and seven as early according to their time of synthesis and also according to their synthesis in the presence of actinomycin D following the reversal of a cycloheximide mediated block in protein synthesis. The estimated molecular weights ranged from 28K - 100K. The synthesis of a dominant IE protein of 100K was significantly increased, after the reversal of a cycloheximide block, compared to unenhanced conditions. The synthesis of two other major IE proteins of 96K and 89K were also significantly enhanced by this treatment. The 100K and 89K proteins partitioned with the nuclear, cytoplasmic and cytoskeletal fractions, while the 96K protein partitioned more strongly with the nuclei. These proteins were phosphorylated. The other IE proteins were synthesized in lesser amounts. The major early proteins, which had molecular weights of 39K and 36K, were also phosphorylated and were exclusively nucleus-associated. A number of the IE and early proteins had affinity for native and denatured DNA-cellulose. The same major IE and early proteins were identified in nonpermissively infected J774A.1 macrophage cells. Although 0.6% of these cells became permissively infected with MCMV and the rest appeared to be nonpermissively infected, viral DNA and late protein synthesis was not detected. The major difference between the proteins produced in 3T3-L1 cells and J774A.1 cells was the affinity of the 96K protein for denatured DNA-cellulose, which was only observed when the protein was synthesized in J774A.1 cells. The main IE and early MCMV induced proteins were also synthesized in nonpermissively infected human fibroblast cells. The only difference between the proteins produced in these cells and 3T3-L1 cells was that the 100K IE protein appeared to have a greater nuclear-affinity, when produced in the human fibroblasts, than was found when synthesized in infected 3T3-L1 cells. In conclusion, a larger number of IE and early MCMV-induced proteins were identified in infected cells than had been previously characterized. There was no evidence of restricted MCMV gene expression occurring in two different cell types that were nonpermissively infected. This appeared to indicate that, in the nonpermissive experiments described, MCMV replication was inhibited at the stage of viral DNA synthesis.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

5

Scott, Felicia Yi Xia. "Controlled Hybrid Material Synthesis using Synthetic Biology." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/86147.

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The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function. Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering.
Ph. D.

6

Schwartz, Anne. "Characterization of normal and androgen resistant-genital skin fibroblasts using high-resolution two-dimensional gel electrophoresis." Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=63378.

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7

Zhang, Yinfeng, and 张银凤. "Protein chemical synthesis by serine and threonine ligation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/202359.

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Landmark advances in the field of synthetic protein chemistry have enabled the preparation of complex, homogeneous proteins, including those that carry specific posttranslational modifications (PTMs). In addition, chemical synthesis will allow one to incorporate unnatural elements to generate new biologics with altered properties and functions. Native chemical ligation (NCL) is a milestone in the chemical synthesis of proteins (Kent et al., Science, 1994, 266, 776-779), in which a C-terminal peptide thioester and an N-terminal cysteine (Cys)-containing peptide-both in side-chain unprotected forms-are selectively coupled to generate a natural peptidic linkage at the site of ligation. This method requires a cysteine at the optimal convergent ligation site. However, Cys is one of the least abundant amino acids in natural proteins. Therefore, the development of new ligation methods at other amino acids will be necessary and important in this regard. Along these lines, our laboratory has developed a novel thiol-independent approach-serine/threonine ligation (STL). It uses the N-terminal serine or threonine of a peptide segment to chemoselectively react with another peptide segment with a C-terminal salicylaldehyde ester to form an N,O-benzylidene acetal linked product, followed by acidolysis to afford the final product at the natural Ser/Thr site. To extend the application of STL in chemical protein synthesis, we have developed a robust method for the preparation of peptide salicylaldehyde esters via Fmoc-based solid phase peptide synthesis. Furthermore, we have successfully applied this ligation method in the convergent synthesis of peptide drugs of significant therapeutic importance, including Teriparatide (Forteo), Corticorelin (oCRH), Exenatide (Byetta) and Tesamorelin (hGHRH). Of significance, we have demonstrated the effectiveness of our STL in the assembly of a more complex target of biological interest: human erythrocyte acylphosphatase (~ 11 kDa). In summary, we have developed a new serine/threonine ligation, which can be effectively used to synthesize peptides and proteins. As there are countless serine and threonine residues in natural proteins, particularly those carrying posttranslational modifications, this method is anticipated to offer new opportunities in synthetic protein chemistry and chemical biology.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

8

Johnston, Julie Catherine. "In vitro translation of cucumber necrosis virus RNA." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/28969.

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The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein.
Land and Food Systems, Faculty of
Graduate

9

Sun, Xiaojiao. "High Affinity Synthetic Molecular Binders for Proteins : Design, Synthesis and Evaluation." Doctoral thesis, Uppsala universitet, Fysikalisk-organisk kemi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-183203.

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This thesis describes the design and synthesis of small molecule derivatives and their polypeptide conjugates as high affinity binders for proteins: the D-dimer protein (D-dimer), a biomarker for diagnosis of thromboembolic diseases; human myeloperoxidase (MPO), a biomarker for cardiovascular diseases; and chitinases, potential targets for asthma therapy. The interactions between the synthetic binder molecules and those proteins were evaluated by surface plasmon resonance (SPR) biosensor analysis and fluorescence spectroscopy. Competition SPR experiments or other methods proved that the small molecule components of the binder molecules were critical for binding and specifically bound to the original binding site of small molecules. The binder molecules consisted of a 42-residue helix-loop-helix polypeptide conjugated to a small molecule via aliphatic spacers of suitable length. The small molecules could be any type of moderately binding structure. In the binder development for the D-dimer, the tetrapeptide GPRP with a dissociation constant Kd of 25 μM was used and the affinity of 4C15L8GPRP obtained was estimated to be approximately 3 nM. In the binder development for MPO, salicylhydroxamic acid (SHA) with Kd of 2 μM was used and the affinity of 4C37L34C11SHA obtained was estimated to be approximately 0.4 nM. In the binder development for chitinases, a theobromine derivative (pentoxifylline) with a Kd of 43±10 μM was used and the affinity of 4C37L34-P obtained was estimated to be considerably higher than that of pentoxifylline. The binder molecules were identified from a 16-membered pool of candidates obtained by conjugating the small molecules to each member of a set of 16 designed polypeptides. The affinities were greatly enhanced by 2-3 orders of magnitude, compared to the small molecule. The polypeptides did not bind to the proteins with measurable affinities. The discovery of these new synthetic binders for protein targets can pave the way to diagnostic tests in vivo or in vitro, independent of antibodies.

10

Kim, Daniel. "Characterization of the Mata pre-." Scholarly Commons, 2009. https://scholarlycommons.pacific.edu/uop_etds/738.

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Книги з теми "Proteins Synthesis":

1

H, Nierhaus Knud, and Wilson Daniel N, eds. Protein synthesis and ribosome structure: Translating the genome. Weinheim: Wiley-VCH, 2004.

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2

E, Esterhouse Toma, and Petrinos Lado B, eds. Protein biosynthesis. New York: Nova Science, 2008.

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3

Lloyd-Williams, Paul. Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press, 1997.

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4

1958-, Martin Robin, ed. Protein synthesis: Methods and protocols. Totowa, N.J: Humana Press, 1998.

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5

Belgium) International Solvay Conference on Chemistry (23rd 2013 Brussels. New chemistry and new opportunities from the expanding protein universe: 23rd International Solvay Conference on Chemistry, Hotel Métropole, Brussels, 16-19 October 2013. Edited by Wüthrich Kurt editor. Hackensack, New Jersey: World Scientific, 2014.

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6

M, Blow D., Fersht Alan 1943-, Winter G, and Royal Society (Great Britain), eds. Design, construction, and properties of novel protein molecules: Proceedings of a Royal Society discussion meeting, held on 5 and 6 June 1985. London: Royal Society, 1986.

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7

Symposium on Molecular Mechanisms in Protein Synthesis (1983 Emirgân, Turkey). Mechanisms of protein synthesis: Structure-function relations, control mechanisms, and evolutionary aspects : Proceedings of the Symposium on Molecular Mechanisms in Protein Synthesis held at Beyaz Köşk, Emigran, Bosphorus, Istanbul. Berlin: Springer-Verlag, 1985.

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8

Giovanni, Cesareni, ed. Modular protein domains. Weinheim: Wiley-VCH, 2005.

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9

L, Hatfield Dolph, Lee Byeong J, and Pirtle Robert M, eds. Transfer RNA in protein synthesis. Boca Raton: CRC Press, 1992.

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10

Hengge-Aronis, Regine. Studies of secretion of periplasmic proteins in Escherichia coli. Konstanz: Hartung-Gorre, 1986.

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Частини книг з теми "Proteins Synthesis":

1

Buxbaum, Engelbert. "Synthesis of Peptides." In Biophysical Chemistry of Proteins, 289–90. Boston, MA: Springer US, 2010. http://dx.doi.org/10.1007/978-1-4419-7251-4_31.

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2

Kermode, Allison R., and J. Derek Bewley. "Synthesis, processing and deposition of seed proteins: The pathway of protein synthesis and deposition in the cell." In Seed Proteins, 807–41. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4431-5_34.

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3

Houghten, Richard A., Sarah R. Hoffmann, Mairead K. Bray, Nicole Frizzell, J. M. Ostresh, Suzanne M. Pratt, and John Sitarik. "Simultaneous Multiple Peptide Synthesis: The Rapid Preparation of Large Numbers of Peptides." In Proteins, 463–70. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_46.

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4

Rivier, J., R. Galyean, W. Woo, D. Karr, T. Richmond, and J. Spiess. "The Symposium Test Peptide (STP): Synthesis and Characterization of a Model Peptide." In Proteins, 789–95. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_81.

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5

Liu, Lei, Sam Danishefsky, and David Crich. "Chemical Synthesis of Proteins." In Organic Chemistry - Breakthroughs and Perspectives, 221–45. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2012. http://dx.doi.org/10.1002/9783527664801.ch6.

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6

Frigerio, Lorenzo, and Lynne M. Roberts. "The Synthesis of Ricinus communis Lectins." In Toxic Plant Proteins, 191–205. Berlin, Heidelberg: Springer Berlin Heidelberg, 2010. http://dx.doi.org/10.1007/978-3-642-12176-0_10.

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7

Buntru, Matthias, Simon Vogel, Ricarda Finnern, and Stefan Schillberg. "Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins." In Recombinant Proteins in Plants, 113–24. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2241-4_8.

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AbstractPlant cell-free lysates contain all the cellular components of the protein biosynthesis machinery, providing an alternative to intact plant cells, tissues, and whole plants for the production of recombinant proteins. Cell-free lysates achieve rapid protein production (within hours or days) and allow the synthesis of proteins that are cytotoxic or unstable in living cells. The open nature of cell-free lysates and their homogeneous and reproducible performance is ideal for protein production, especially for screening applications, allowing the direct addition of nucleic acid templates encoding proteins of interest, as well as other components such as enzyme substrates, chaperones, artificial amino acids, or labeling molecules. Here we describe procedures for the production of recombinant proteins in the ALiCE (Almost Living Cell-free Expression) system, a lysate derived from tobacco cell suspension cultures that can be used to manufacture protein products for molecular and biochemical analysis as well as applications in the pharmaceutical industry.

8

Tanoue, Eiichiro. "Proteins in the Sea — Synthesis." In Dynamics and Characterization of Marine Organic Matter, 383–463. Dordrecht: Springer Netherlands, 2000. http://dx.doi.org/10.1007/978-94-017-1319-1_18.

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9

Fernández-Tejada, Alberto, John Brailsford, Qiang Zhang, Jae-Hung Shieh, Malcolm A. S. Moore, and Samuel J. Danishefsky. "Total Synthesis of Glycosylated Proteins." In Protein Ligation and Total Synthesis I, 1–26. Cham: Springer International Publishing, 2014. http://dx.doi.org/10.1007/128_2014_622.

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10

Halvorson, Harlyn O. "The Induced Synthesis of Proteins." In Advances in Enzymology - and Related Areas of Molecular Biology, 99–156. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2006. http://dx.doi.org/10.1002/9780470122679.ch3.

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Тези доповідей конференцій з теми "Proteins Synthesis":

1

Jackson, C. W., N. K. Hutson, and S. A. Steward. "CHANGES IN PROTEIN SYNTHESIS PROFILES OF MEGAKARYOCYTES (MK) DURING MATURATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643545.

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Several key differentiation events occur within the recognizable MK compartment; however, little is known about the macromolecular changes responsible for these events. In this study, protein synthesis profiles of morphologically immature and mature guinea pig MK populations have been analyzed by twodimensional gel electrophoresis after in vivo labeling with 35S-methionine. MK were enriched by a bovine plasma aggregation enrichment procedure (Blood 69:173, 1987) and then fractionated into immature and mature populations based on differences in their respective buoyant densities (Brit. J. Haematol. 64:33, 1986). With this protocol, immature and mature MK populations were obtained in which MK constituted 95% of the cell mass. Ninety percent of the MK in the immature population had basophilic, immature morphology while ≥90% of those in the mature population had acidophilic, mature staining characteristics after Wright's staining. Protease inhibitors were used throughout the isolation procedure. The cells were solubilized and proteins subjected to two-dimensional electrophoresis according to O'Farrell (J. Biol. Chem. 250:4007, 1975). To examine basic proteins, proteins were electrophoresed in the first dimension under nonequilibrium conditions in a pH gradient as described by O'Farrell et al. (Cell 12:1133, 1977). Analyses of fluorograms revealed both qualitative and quantitative differences in synthesis profiles between these two MK populations. Among acidic proteins whose synthesis was readily detected in immature but not mature MK were ones whose MW and pi were respectively: 120K, 6.4; 7OK, 5.9; 70K, 6.9; 65K, 6.8; 55K, 6.2; 55K, 6.0; 53K, 5.8; 53K, 6.5; 52K, 6.7; 50K, 6.8; 41K, 5.5 and 33K, 6.7. Acidic and neutral proteins prominently synthesized in mature but not immature MK were found at MW and PI of: 110K, 5.7; 110K, 5.8 and 80K, 7.2. Basic proteins prominently synthesized in immature but not mature MK were found at MWs of: 110K; 70K; 52K; 48K; 39K and 18K. Basic proteins actively synthesized by mature but not immature MK had MWs of: 83K; 43K and 17K. These findings demonstrate that differences in protein synthesis patterns can be readily detected between immature and mature MK and provide baseline data with which to explore the role of these proteins in MK differentiation

2

Mattern-Schain, Samuel I., Mary-Anne Nguyen, Tayler M. Schimel, James Manuel, Joshua Maraj, Donald Leo, Eric Freeman, Scott Lenaghan, and Stephen A. Sarles. "Totipotent Cellularly-Inspired Materials." In ASME 2019 Conference on Smart Materials, Adaptive Structures and Intelligent Systems. American Society of Mechanical Engineers, 2019. http://dx.doi.org/10.1115/smasis2019-5745.

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Abstract This work draws inspiration from totipotent cellular systems to design smart materials whose compositions and properties can be learned or evolved. Totipotency refers to the inherent genetic potential of a single cell to adapt and produce all types of differentiated cells within an organism. To study this principal and apply it synthetically, tissue-like compartmentalized assemblies are constructed via lipid membrane-separated aqueous droplets in a hydrophobic medium through the droplet interface bilayer (DIB) method. Within our droplets, we explore synthetic totipotency via cell-free reactions including actin polymerization and cell free protein synthesis (CFPS). The transcription and translation of our CFPS reactions are controlled by stimuli-responsive riboswitches (RS). Via this scheme, adaptable material properties and functions are achieved in vitro via protein production from cell-free machinery administered through RS governance. Here, we present thermally or chemically-triggered riboswitches for orthogonal production of representative fluorescent protein products, as well functional proteins. To characterize the material properties of target proteins, we study the formation of polymerized actin shells to stabilize organically-encased droplets and span DIBs. We present a modified protocol for chemically-triggered actin polymerization as well as a thermally triggered actin RS. We characterize theophylline (TP)-triggered production of alpha hemolysin (α-HL) through CFPS and synthesized an organic-soluble trigger that can be sensed from the oil phase by a RS in an aqueous bioreactor droplet. We also demonstrate increased droplet conductivity when CFPS α-HL products are incorporated in DIBs. This interdisciplinary work involves cell culture, gene expression, organic synthesis, vesicle formation, protein quantification, tensiometry, droplet aspiration, microplate fluorescence/absorption experiments, fluorescent microscopy, and electrophysiology. This project is an essential design analysis for creating smart, soft materials using synthetic biology and provides motivation for artificial tissues capable of adapting in response to external stimuli.

3

Rao, Jiajia. "Tuning plant protein for improved functionality and flavor profile: From field to application." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/xqxj4886.

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Recently, plant proteins are gaining in popularity as consumers are looking to rebalance their diet with more plant-based options. As such, there is a need to understand the potential of these plant proteins to deliver nutrition and functionality in various food products. However, plant proteins are still under-utilization in food and beverage industry due to their characteristic beany and off flavor, lower technical functionality, such as limited solubility, and some negative taste attributes. In general, plant protein can be obtained from pulses, cereals and oilseeds. Each plant has a unique protein structure and composition, thus protein extracted from different plant has different functional properties and performance in food. In general, plant contain non-protein components including starch, fiber and oil. High purity of plant protein are often extracted to by removing all abovementioned non-protein components through extraction processing. One would expect different extraction methods can influence protein functionality through denaturation, modification, hydrolysis and cross-linking. This talk begins with the discussion of how different plant variety and processing impact on protein functionality using hemp protein as an example. Selective physical and chemical modification methods (e.g., soluble complexes, pH shift, maillard reaction, phosphorylation) for generating novel protein constructs with preferable solubility, foaming, and emulsification properties, thermal stability, and flavor profiles will be comprehensively discussed by using pea protein isolate (PPI) as a representative. In general, protein solubility could be improved by means of abovementioned three methods. Maillard-driven synthesis of the cross-linked PPI-gum arabic conjugates greatly improved the flavor profile and functionality of PPI. In terms of phosphorylation, sodium hexametaphosphate (SHMP) is a good candidate to form phosphorylated PPI with enhanced functionalities including foaming, emulsifying properties and thermal stability. Our results suggested that protein structure-function researches are valuable in tailoring proteins for specific functional outcomes and expanding the availability of plant proteins.

4

Waart, P. v. d., K. T. Preissner, U. Delvos, and G. Müller-Berghaus. "SIMULTANEOUS PRODUCTION OF ENDOTHELIAL CELL-DERIVED PROTEIN S AND FACTOR V, AND INACTIVATION OF FACTOR Va AT THE ENDOTHELIAL CELL SURFACE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644737.

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Several proteins synthesized and expressed by endothelial cells are involved in the regulation of coagulation. The synthesis and expression of factor V and protein S has been demonstrated in independent studies. The present work evaluates the simultaneous synthesis and expression of bovine factor V and protein S and the effect of endothelial protein S on the inactivation of endothelial factor Va by activated protein C. The accumulation of both proteins in conditioned medium was detected by SDS-PAGE followed by immunoblotting, and their activities were tested by functional assays. The synthesis of protein S and factor V per 105 cells over 24 h amounted up to 2 ng protein S and 440 ng factor V, respectively. The addition of thrombin did not increase the yield of synthesized cofactors. Thrombin did neither proteolyse protein S on endothelial cells nor in a purified system in the presence of thrombomodulin and calcium ions. Factor V was secreted partly in its activated form as evidenced by the appearance of active intermediates with M = 220,000-280,000 on immunoblots as well as by only a three-Fold further activation of factor V/Va following addition of thrombin. The rate constant for the inactivation of factor Va by activated protein C was only two-fold higher for factor Va derived from cells cultured in the presence of vitamin K as compared in the presence of warfarin. For the inactivation of comparable factor Va concentrations in conditioned medium a 10-fold higher and on endothelial cells a 40-fold higher concentration of activated protein C was required to obtain similar inactivation rates of factor Va as compared to a purified system. These results suggest that resting endothelial cells contain a factor V activator, and that a regulatory mechanism is operative on the endothelial cell surface that suppresses the inactivation potential of activated protein C/ protein S.

5

TAM, James P. "Design and Synthesis of Peptide Biologics by Deconstruction of Proteins." In The Twenty-Third American and the Sixth International Peptide Symposium. Prompt Scientific Publishing, 2013. http://dx.doi.org/10.17952/23aps.2013.001.

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6

Macmillan, Derek, and Alison M. Daines. "SEMI-SYNTHESIS OF GLYCOSYLATED PROTEINS AND MIMICS USING CHEMOSELECTIVE LIGATIONS." In XXIst International Carbohydrate Symposium 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.438.

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7

Gaivoronskaya, Irina, and Valenitna Kolpakova. "MATHEMATICAL MODELS FOR THE SYNTHESIS OF PLANT-BASED COMPOSITIONS WITH IMPROVED AMINO ACID COMPOSITION." In GEOLINKS Conference Proceedings. Saima Consult Ltd, 2021. http://dx.doi.org/10.32008/geolinks2021/b1/v3/12.

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The aim of the work was to optimize the process of obtaining multicomponent protein compositions with high biological value and higher functional properties than the original vegetable protein products. Was realized studies to obtain biocomposites on the base of pea protein-oat protein and pea protein-rice protein. Developed composites were enriched with all limited amino acids. For each of the essential amino acids, the amino acid score was 100% and higher. Protein products used in these compositions are not in major allergen list, which allows to use these compositions in allergen-free products and specialized nutrition. To determine biosynthesis parameters for compositions from pea protein and various protein concentrates with the use of transglutaminase enzyme, was studied effect of concentration and exposition time on the amount of amino nitrogen released during the reaction. Decreasing of amino nitrogen in the medium indicated the occurrence of a protein synthesis reaction with the formation of new covalent bonds. Were determined optimal parameters of reaction: the hydromodule, the exposure time, the concentration of EP of the preparation, were obtained mathematical models. Studies on the functional properties of composites, the physicochemical properties of the proteins that make up their composition, and structural features will make it possible to determine the uses in the manufacture of food products based on their ability to bind fat, water, form foam, gels, and etc.

8

Banneyake, B. M. R. U., and Debjyoti Banerjee. "Microfluidic Device for Synthesis of Lipid Bi-Layers." In ASME 2008 Fluids Engineering Division Summer Meeting collocated with the Heat Transfer, Energy Sustainability, and 3rd Energy Nanotechnology Conferences. ASMEDC, 2008. http://dx.doi.org/10.1115/fedsm2008-55219.

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Lipid bi-layers are ubiquitous components of biological cells — and are found in variety of cell components ranging from cell membranes to membranes of organelles inside the cells. In biological membranes, lipid bi-layer membranes carry membrane proteins, which serve as single channel nanopores that are used to study transport of proteins and characterize the properties of proteins. However, lipid bi-layers have very short half lives, which are usually less than an hour. The lipid bi-layers are usually obtained by physico-chemical interactions between a lipid containing organic solvent, an aqueous buffer solution and a hydrophobic surface. We have developed a continuous flow through microfluidic device using pressure driven flow (by means of a tandem syringe pump system) for synthesis of lipid bi-layers. The microfluidic device consists of two glass substrates with micro-channels and microchambers microfabricated using photolithography and wet glass etching. The microchannels in each substrate is in the form of “+” shape and form a mirror image of each other. A Teflon sheet (∼200 microns thickness) is sandwiched between the glass substrates with a ∼200 microns diameter hole etched in the center to communicate with the two sets of microchannels. A lipid solution in an organic solvent (Pentane) and KCl buffer solution are alternately flown through the legs of the microchannel. The conductivity of the buffer is monitored using a current amplifier. The formation of the lipid bi-layer is confirmed by monitoring the resistivity and the impedance to high frequency electrical oscillations. The flow rate in the microfluidic device is optimized to obtain the lipid bi-layer.

9

Verhulst, Anja, Veerle P. Persy, and Marc E. De Broe. "SYNTHESIS, SECRETION AND LOCALIZATION OF OSTEOPONTIN IN THE HUMAN NEPHRON." In 3rd International Conference on Osteopontin and SIBLING (Small Integrin-Binding Ligand, N-linked Glycoprotein) Proteins, 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.331.

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10

Teng, Weibing, Joseph Cappello, and Xiaoyi Wu. "Viscoelastic Properties of Genetically Engineered Silk-Elastin-Like Protein Polymers." In ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-192252.

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Genetic engineering of protein-based materials provides material scientists with high levels of control in material microstructures, properties, and functions [1]. For example, multi-block protein copolymers in which individual block may possess distinct mechanical or biological properties have been biosynthesized [2, 3]. Polypeptide sequences derived from well-studied structural proteins (e.g., collagen, silk, elastin) are often used as motifs in the design and synthesis of new protein-based material, in which new functional groups may be incorporated. In this fashion, we have produced a series of silk-elastin-like proteins (SELPs) consisting of polypeptide sequences derived from silk of superior mechanical strength and elastin that is extremely durable and resilient [2, 4]. Notably, the silk-like blocks are capable of crystallizing to form virtual cross-links between elastin-mimetic sequences, which, in turn, lower the crystallinity of the silk-like blocks and thus enhance the solubility of SELPs. Consequently, SELPs may be fabricated into useful structures for biomedical applications, including drug delivery. In this study, we will characterize viscoelastic properties of SELPs, which are particularly relevant to tissue engineering applications.

Звіти організацій з теми "Proteins Synthesis":

1

Desai, Holly E. Synthesis and Structural Characterization of Reflectin Proteins. Fort Belvoir, VA: Defense Technical Information Center, February 2012. http://dx.doi.org/10.21236/ada563720.

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2

Deutsch, Christopher. Discovery and Characterization of the Proteins Involved in the Synthesis of N⁶-Threonylcarbamoyl Adenosine, a Nucleoside Modification of tRNA. Portland State University Library, January 2000. http://dx.doi.org/10.15760/etd.3075.

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3

Barash, Itamar, and Robert E. Rhoads. Translational Mechanisms that Govern Milk Protein Levels and Composition. United States Department of Agriculture, November 2004. http://dx.doi.org/10.32747/2004.7586474.bard.

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Original objectives: The long term objective of the project is to achieve higher content of protein in the milk of ruminants by modulating the translational machinery in the mammary gland. The first specific aim of the BARD proposal was to characterize responsiveness of various experimental systems to combination of lactogenic hormones and amino acids with particular emphasis on discrimination between the control of total protein synthesis and milk protein synthesis. Based on the results, we planned to proceed by characterizing the stage of protein synthesis in which the stimulation by lactogenic hormones and amino acid occur and finally we proposed to identify which components of the translation machinery are modified. Background to the topic: Milk protein is the most valuable component in milk, both for direct human consumption and for manufacturing cheese and other protein-based products. Attempts to augment protein content by the traditional methods of genetic selection and improved nutritional regimes have failed. The proposal was based on recent results suggesting that the limiting factor for augmenting protein synthesis in the bovine mammary gland is the efficiency of converting amino acids to milk proteins. Major conclusions, solutions, achievements: Insulin and prolactin synergistically stimulate â-casein mRNA translation by cytoplasmatic polyadenylation. The interaction between insulin and prolactin was demonstrated two decades ago as crucial for milk-protein synthesis, but the molecular mechanisms involved were not elucidated. We found in differentiated CID 9 mouse mammary epithelial cells line that insulin and prolactin synergistically increases the rate of milk protein mRNA translation. We focused on â-casein, the major milk protein, and found that the increase in â-casein mRNA translation was reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the PI3K, mTOR, and MAPK pathways blocked insulin-stimulated total protein and â-casein synthesis but not the synergistic stimulation. Conversely, cordycepin, a polyadenylation inhibitor, abolished synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of â-casein mRNA progressively increased over 30 min of treatment with insulin plus prolactin. The 3’-untranslated region of â-casein mRNA was found to contain a cytoplasmic polyadenylation element (CPE), and in reporter constructs, this was sufficient for the translational enhancement and mRNA-specific polyadenylation. Furthermore, insulin and prolactin stimulated phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) but did not increase cytoplasmic polyadenylation.

4

Barash, Itamar, and Robert Rhoads. Translational Mechanisms Governing Milk Protein Levels and Composition. United States Department of Agriculture, 2006. http://dx.doi.org/10.32747/2006.7696526.bard.

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Original objectives: The long-term goal of the research is to achieve higher protein content in the milk of ruminants by modulating the translational apparatus of the mammary gland genetically, nutritionally, or pharmacologically. The short-term objectives are to obtain a better understanding of 1) the role of amino acids (AA) as regulators of translation in bovine and mouse mammary epithelial cells and 2) the mechanism responsible for the synergistic enhancement of milk-protein mRNA polyadenylation by insulin and prolactin. Background of the topic: In many cell types and tissues, individual AA affect a signaling pathway which parallels the insulin pathway to modulate rates and levels of protein synthesis. Diverse nutritional and hormonal conditions are funneled to mTOR, a multidomain serine/threonine kinase that regulates a number of components in the initiation and elongation stages of translation. The mechanism by which AA signal mTOR is largely unknown. During the current grant period, we have studied the effect of essential AA on mechanisms involved in protein synthesis in differentiated mammary epithelial cells cultured under lactogenic conditions. We also studied lactogenic hormone regulation of milk protein synthesis in differentiated mammary epithelial cells. In the first BARD grant (2000-03), we discovered a novel mechanism for mRNA-specific hormone-regulated translation, namely, that the combination of insulin plus prolactin causes cytoplasmic polyadenylation of milk protein mRNAs, which leads to their efficient translation. In the current BARD grant, we have pursued the signaling pathways of this novel hormone action. Major conclusions/solutions/achievements: The positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediates the synthesis rates of total and specific milk proteins in mammary epithelial cells. The current in vitro study revealed cryptic negative effects of Lys, His, and Thr on cellular mechanisms regulating translation initiation and protein synthesis in mammary epithelial cells that could not be detected by conventional in vivo analyses. We also showed that a signaling pathway involving Jak2 and Stat5, previously shown to lead from the prolactin receptor to transcription of milk protein genes, is also used for cytoplasmic polyadenylation of milk protein mRNAs, thereby stabilizing these mRNAs and activating them for translation. Implications: In vivo, plasma AA levels are affected by nutritional and hormonal effects as well as by conditions of exercise and stress. The amplitude in plasma AA levels resembles that applied in the current in vitro study. Thus, by changing plasma AA levels in the epithelial cell microenvironment or by sensitizing the mTOR pathway to their presence, it should be possible to modulate the rate of milk protein synthesis. Furthermore, knowledge that phosphorylation of Stat5 is required for enhanced milk protein synthesis in response to lactogenic opens the possibility for pharmacologic approaches to increase the phosphorylation of Stat5 and, thereby, milk protein production.

5

Amir, Rachel, David J. Oliver, Gad Galili, and Jacline V. Shanks. The Role of Cysteine Partitioning into Glutathione and Methionine Synthesis During Normal and Stress Conditions. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7699850.bard.

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The objective of this research is to study the nature of the competition for cysteine (Cys), the first organic sulfur-containing compound, between its two main metabolites, glutathione (GSH) and methionine (Met). GSH plays a central role in protecting plants during various stresses, while Met, an essential amino acid, regulates essential processes and metabolites in plant cells through its metabolite S-adenosyl-Met. Our results, which are based on flux analysis and measurements of Met- metabolites, show that the flux towards Met synthesis is high during non-stress conditions, however the flux is significantly reduced under stress conditions, when there is high synthesis of GSH. Under oxidative stress the expression level of the regulatory enzyme of Met synthesis, cystathionine g-synthase (CGS) was reduced. By using three different systems, we have found that that GSH down regulates the expression level of CGS, thus reducing Met synthesis. We have found that this regulation occurs at the post-transcriptional level, and further studies have shown that it occurs at post-translationaly. To reveal how oxidative stress affects the flux towards Met and GSH, flux analysis was performed. We have found that the level of Met is significantly reduced, while the level of glutathione significantly increases during stress. Under stress conditions most of the glutathione is converted from GSH to GSSG (the oxidised form of glutathione). These results suggest that under normal growth conditions, Cys is channelled towards both pathways to support GSH accumulation and the synthesis of growth-essential Met metabolites. However, during oxidative stress, when a high level of GSH is required to protect the plants, the levels of GSH increase while those of CGS are reduced. This reduction leaves more Cys available for GSH synthesis under stress conditions. In addition we have also studied the effects of high GSH level on the transcriptome profile. The analysis revealed that GSH affects the expression level of many major genes coding to enzymes or proteins associated with photosynthesis, starch degradation, hormone metabolism (especially genes associated with jasmonate), biotic stress (especially genes associated with PR-proteins), cytochrome P450 genes, regulation of transcription and signaling (especially genes associated with receptor kinases and calcium). These results suggest that indeed GSH levels affect different pathways and metabolites in plants.

6

Christopher, David A., and Avihai Danon. Plant Adaptation to Light Stress: Genetic Regulatory Mechanisms. United States Department of Agriculture, May 2004. http://dx.doi.org/10.32747/2004.7586534.bard.

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Original Objectives: 1. Purify and biochemically characterize RB60 orthologs in higher plant chloroplasts; 2. Clone the gene(s) encoding plant RB60 orthologs and determine their structure and expression; 3. Manipulate the expression of RB60; 4. Assay the effects of altered RB60 expression on thylakoid biogenesis and photosynthetic function in plants exposed to different light conditions. In addition, we also examined the gene structure and expression of RB60 orthologs in the non-vascular plant, Physcomitrella patens and cloned the poly(A)-binding protein orthologue (43 kDa RB47-like protein). This protein is believed to a partner that interacts with RB60 to bind to the psbA5' UTR. Thus, to obtain a comprehensive view of RB60 function requires analysis of its biochemical partners such as RB43. Background & Achievements: High levels of sunlight reduce photosynthesis in plants by damaging the photo system II reaction center (PSII) subunits, such as D1 (encoded by the chloroplast tpsbAgene). When the rate of D1 synthesis is less than the rate of photo damage, photo inhibition occurs and plant growth is decreased. Plants use light-activated translation and enhanced psbAmRNA stability to maintain D1 synthesis and replace the photo damaged 01. Despite the importance to photosynthetic capacity, these mechanisms are poorly understood in plants. One intriguing model derived from the algal chloroplast system, Chlamydomonas, implicates the role of three proteins (RB60, RB47, RB38) that bind to the psbAmRNA 5' untranslated leader (5' UTR) in the light to activate translation or enhance mRNA stability. RB60 is the key enzyme, protein D1sulfide isomerase (Pill), that regulates the psbA-RN :Binding proteins (RB's) by way of light-mediated redox potentials generated by the photosystems. However, proteins with these functions have not been described from higher plants. We provided compelling evidence for the existence of RB60, RB47 and RB38 orthologs in the vascular plant, Arabidopsis. Using gel mobility shift, Rnase protection and UV-crosslinking assays, we have shown that a dithiol redox mechanism which resembles a Pill (RB60) activity regulates the interaction of 43- and 30-kDa proteins with a thermolabile stem-loop in the 5' UTR of the psbAmRNA from Arabidopsis. We discovered, in Arabidopsis, the PD1 gene family consists of II members that differ in polypeptide length from 361 to 566 amino acids, presence of signal peptides, KDEL motifs, and the number and positions of thioredoxin domains. PD1's catalyze the reversible formation an disomerization of disulfide bonds necessary for the proper folding, assembly, activity, and secretion of numerous enzymes and structural proteins. PD1's have also evolved novel cellular redox functions, as single enzymes and as subunits of protein complexes in organelles. We provide evidence that at least one Pill is localized to the chloroplast. We have used PDI-specific polyclonal and monoclonal antisera to characterize the PD1 (55 kDa) in the chloroplast that is unevenly distributed between the stroma and pellet (containing membranes, DNA, polysomes, starch), being three-fold more abundant in the pellet phase. PD1-55 levels increase with light intensity and it assembles into a high molecular weight complex of ~230 kDa as determined on native blue gels. In vitro translation of all 11 different Pill's followed by microsomal membrane processing reactions were used to differentiate among PD1's localized in the endoplasmic reticulum or other organelles. These results will provide.1e insights into redox regulatory mechanisms involved in adaptation of the photosynthetic apparatus to light stress. Elucidating the genetic mechanisms and factors regulating chloroplast photosynthetic genes is important for developing strategies to improve photosynthetic efficiency, crop productivity and adaptation to high light environments.

7

Lapidot, Moshe, and Vitaly Citovsky. molecular mechanism for the Tomato yellow leaf curl virus resistance at the ty-5 locus. United States Department of Agriculture, January 2016. http://dx.doi.org/10.32747/2016.7604274.bard.

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Tomato yellow leaf curl virus (TYLCV) is a major pathogen of tomato that causes extensive crop loss worldwide, including the US and Israel. Genetic resistance in the host plant is considered highly effective in the defense against viral infection in the field. Thus, the best way to reduce yield losses due to TYLCV is by breeding tomatoes resistant or tolerant to the virus. To date, only six major TYLCV-resistance loci, termed Ty-1 to Ty-6, have been characterized and mapped to the tomato genome. Among tomato TYLCV-resistant lines containing these loci, we have identified a major recessive quantitative trait locus (QTL) that was mapped to chromosome 4 and designated ty-5. Recently, we identified the gene responsible for the TYLCV resistance at the ty-5 locus as the tomato homolog of the gene encoding messenger RNA surveillance factor Pelota (Pelo). A single amino acid change in the protein is responsible for the resistant phenotype. Pelo is known to participate in the ribosome-recycling phase of protein biosynthesis. Our hypothesis was that the resistant allele of Pelo is a “loss-of-function” mutant, and inhibits or slows-down ribosome recycling. This will negatively affect viral (as well as host-plant) protein synthesis, which may result in slower infection progression. Hence we have proposed the following research objectives: Aim 1: The effect of Pelota on translation of TYLCV proteins: The goal of this objective is to test the effect Pelota may or may not have upon translation of TYLCV proteins following infection of a resistant host. Aim 2: Identify and characterize Pelota cellular localization and interaction with TYLCV proteins: The goal of this objective is to characterize the cellular localization of both Pelota alleles, the TYLCV-resistant and the susceptible allele, to see whether this localization changes following TYLCV infection, and to find out which TYLCV protein interacts with Pelota. Our results demonstrate that upon TYLCV-infection the resistant allele of pelota has a negative effect on viral replication and RNA transcription. It is also shown that pelota interacts with the viral C1 protein, which is the only viral protein essential for TYLCV replication. Following subcellular localization of C1 and Pelota it was found that both protein localize to the same subcellular compartments. This research is innovative and potentially transformative because the role of Peloin plant virus resistance is novel, and understanding its mechanism will lay the foundation for designing new antiviral protection strategies that target translation of viral proteins. BARD Report - Project 4953 Page 2

8

Grafi, Gideon, and Brian Larkins. Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575285.bard.

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The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.

9

Blum, Abraham, Henry T. Nguyen, and N. Y. Klueva. The Genetics of Heat Shock Proteins in Wheat in Relation to Heat Tolerance and Yield. United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568105.bard.

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Fifty six diverse spring wheat cultivars were evaluated for genetic variation and heritability for thermotolerance in terms of cell-membrane stability (CMS) and triphenyl tetrazolium chloride (TTC) reduction. The most divergent cultivars for thermotolerance (Danbata-tolerant and Nacozari-susceptible) were crossed to develop an F8 random onbred line (RIL) population. This population was evaluated for co-segragation in CMS, yield under heat stress and HSP accumulation. Further studies of thermotolerance in relations to HSP and the expression of heterosis for growth under heat stress were performed with F1 hybrids of wheat and their parental cultivars. CMS in 95 RILs ranged from 76.5% to 22.4% with 71.5% and 31.3% in Danbata and Nacozari, respectively. The population segregated with a normal distribution across the full range of the parental values. Yield and biomass under non-stress conditions during the normal winter season at Bet Dagan dit not differ between the two parental cultivar, but the range of segregation for these traits in 138 RILs was very high and distinctly transgressive with a CV of 35.3% and 42.4% among lines for biomass and yield, respectively. Mean biomass and yield of the population was reduced about twofold when grown under the hot summer conditions (irrigated) at Bet Dagan. Segregation for biomass and yield was decreased relative to the normal winter conditions with CV of 20.2% and 23.3% among lines for biomass and yield, respectively. However, contrary to non-stress conditions, the parental cultivars differed about twofold in biomass and yield under heat stress and the population segregated with normal distribution across the full range of this difference. CMS was highly and positively correlated across 79 RILs with biomass (r=0.62**) and yield (r=0.58**) under heat stress. No such correlation was obtained under the normal winter conditions. All RILs expressed a set of HSPs under heat shock (37oC for 2 h). No variation was detected among RILs in high molecular weight HSP isoforms and they were similar to the patterns of the parental cultivars. There was a surprisingly low variability in low molecular weight HSP isoforms. Only one low molecular weight and Nacozari-specific HSP isoform (belonging to HSP 16.9 family) appeared to segregate among all RILs, but it was not quantitatively correlated with any parameter of plant production under heat stress or with CMS in this population. It is concluded that this Danbata/Nacozari F8 RIL population co-segregated well for thermotolerance and yield under heat stress and that CMS could predict the relative productivity of lines under chronic heat stress. Regretfully this population did not express meaningful variability for HSP accumulation under heat shock and therefore no role could be seen for HSP in the heat tolerance of this population. In the study of seven F1 hybrids and their parent cultivars it was found that heterosis (superiority of the F1 over the best parent) for CMs was generally lower than that for growth under heat stress. Hybrids varied in the rate of heterosis for growth at normal (15o/25o) and at high (25o/35o) temperatures. In certain hybrids heterosis for growth significantly increased at high temperature as compared with normal temperature, suggesting temperature-dependent heterosis. Generally, under normal temperature, only limited qualitative variation was detected in the patterns of protein synthesis in four wheat hybrids and their parents. However, a singular protein (C47/5.88) was specifically expressed only in the most heterotic hybrid at normal temperature but not in its parent cultivars. Parental cultivars were significantly different in the sets of synthesized HSP at 37o. No qualitative changes in the patterns of protein expression under heat stress were correlated with heterosis. However, a quantitative increase in certain low molecular weight HSP (mainly H14/5.5 and H14.5.6, belonging to the HSP16.9 family) was positively associated with greater heterosis for growth at high temperature. None of these proteins were correlated with CMS across hybrids. These results support the concept of temperature-dependent heterosis for growth and a possible role for HSP 16.9 family in this respect. Finally, when all experiments are viewed together, it is encouraging to find that genetic variation in wheat yield under chronic heat stress is associated with and well predicted by CMS as an assay of thermotolerance. On the other hand the results for HSP are elusive. While very low genetic variation was expressed for HSP in the RIL population, a unique low molecular weight HSP (of the HSP 16.9 family) could be associated with temperature dependant heterosis for growth.

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Karen S. Browning. Protein Synthesis Initiation Factors: Phosphorylation and Regulation. Office of Scientific and Technical Information (OSTI), June 2009. http://dx.doi.org/10.2172/956983.

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