title: Cell-free protein production and labeling protocol for NMR-based structural proteomics.
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.
authors.affiliation: Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
authors.forename: Dmitriy A
authors.initials: DA
authors.lastname: Vinarov
authors.forename: Betsy L
authors.initials: BL
authors.lastname: Lytle
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.forename: Ejan M
authors.initials: EM
authors.lastname: Tyler
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
authors.forename: John L
authors.initials: JL
authors.lastname: Markley
mesh_terms: D029681:Arabidopsis Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D002474:Cell-Free System / Q000378:metabolism*; D003198:Computer Simulation; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D020543:Proteome