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Nat Methods. 2004 Nov;1(2):149-53. Epub 2004 Oct 21.

Cell-free protein production and labeling protocol for NMR-based structural proteomics.

Author information

1
Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.

Abstract

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.

PMID:
15782178
DOI:
10.1038/nmeth716
[Indexed for MEDLINE]

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