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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

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