Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods 2004 Nov;1(2):149-53
Date
03/23/2005Pubmed ID
15782178DOI
10.1038/nmeth716Scopus ID
2-s2.0-14344256801 (requires institutional sign-in at Scopus site) 122 CitationsAbstract
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.
Author List
Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JLAuthors
Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of WisconsinBrian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Arabidopsis ProteinsCarbon Isotopes
Cell-Free System
Computer Simulation
Gene Expression Profiling
Gene Expression Regulation, Plant
Isotope Labeling
Magnetic Resonance Spectroscopy
Models, Chemical
Nitrogen Isotopes
Protein Conformation
Proteome
Proteomics
Sequence Analysis, Protein
Structure-Activity Relationship
