1se9
From Proteopedia
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==Overview== | ==Overview== | ||
| - | Structural proteomics requires robust, scalable methods. Here we describe | + | Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods. To illustrate this platform, we describe its application to a specific target (At3g01050.1) from Arabidopsis thaliana. After cloning the target gene into a specialized plasmid, we carry out a small-scale (50 mul) in vitro sequential transcription and translation trial to ascertain the level of protein production and solubility. Next, we prepare mRNA for use in a 4-ml semicontinuous cell-free translation reaction to incorporate (15)N-labeled amino acids into a protein sample that we purify and test for suitability for NMR structural analysis. We then repeat the cell-free approach with (13)C,(15)N-labeled amino acids to prepare a doubly labeled sample. The three-dimensional (3D) structure of At3g01050.1 shows that this protein is an unusual member of the beta-grasp protein family. |
==About this Structure== | ==About this Structure== | ||
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[[Category: Arabidopsis thaliana]] | [[Category: Arabidopsis thaliana]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: CESG, Center | + | [[Category: CESG, Center for Eukaryotic Structural Genomics.]] |
| - | [[Category: Lytle, B | + | [[Category: Lytle, B L.]] |
| - | [[Category: Peterson, F | + | [[Category: Peterson, F C.]] |
| - | [[Category: Volkman, B | + | [[Category: Volkman, B F.]] |
[[Category: cell-free]] | [[Category: cell-free]] | ||
[[Category: center for eukaryotic structural genomics]] | [[Category: center for eukaryotic structural genomics]] | ||
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[[Category: wheat germ]] | [[Category: wheat germ]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:00:36 2008'' |
Revision as of 13:00, 21 February 2008
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Structure of At3g01050, a ubiquitin-fold protein from Arabidopsis thaliana
Overview
Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods. To illustrate this platform, we describe its application to a specific target (At3g01050.1) from Arabidopsis thaliana. After cloning the target gene into a specialized plasmid, we carry out a small-scale (50 mul) in vitro sequential transcription and translation trial to ascertain the level of protein production and solubility. Next, we prepare mRNA for use in a 4-ml semicontinuous cell-free translation reaction to incorporate (15)N-labeled amino acids into a protein sample that we purify and test for suitability for NMR structural analysis. We then repeat the cell-free approach with (13)C,(15)N-labeled amino acids to prepare a doubly labeled sample. The three-dimensional (3D) structure of At3g01050.1 shows that this protein is an unusual member of the beta-grasp protein family.
About this Structure
1SE9 is a Single protein structure of sequence from Arabidopsis thaliana. Full crystallographic information is available from OCA.
Reference
Cell-free protein production and labeling protocol for NMR-based structural proteomics., Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL, Nat Methods. 2004 Nov;1(2):149-53. Epub 2004 Oct 21. PMID:15782178
Page seeded by OCA on Thu Feb 21 15:00:36 2008
Categories: Arabidopsis thaliana | Single protein | CESG, Center for Eukaryotic Structural Genomics. | Lytle, B L. | Peterson, F C. | Volkman, B F. | Cell-free | Center for eukaryotic structural genomics | Cesg | Plant protein | Protein structure initiative | Psi | Structural genomics | Ubiquitin-like | Wheat germ
