title: Wheat germ cell-free platform for eukaryotic protein production.
abstract: We describe a platform that utilizes wheat germ cell-free technology to produce protein samples for NMR structure determinations. In the first stage, cloned DNA molecules coding for proteins of interest are transcribed and translated on a small scale (25 microL) to determine levels of protein expression and solubility. The amount of protein produced (typically 2-10 microg) is sufficient to be visualized by polyacrylamide gel electrophoresis. The fraction of soluble protein is estimated by comparing gel scans of total protein and soluble protein. Targets that pass this first screen by exhibiting high protein production and solubility move to the second stage. In the second stage, the DNA is transcribed on a larger scale, and labeled proteins are produced by incorporation of [(15)N]-labeled amino acids in a 4 mL translation reaction that typically produces 1-3 mg of protein. The [(15)N]-labeled proteins are screened by (1)H-(15)N correlated NMR spectroscopy to determine whether the protein is a good candidate for solution structure determination. Targets that pass this second screen are then translated in a medium containing amino acids doubly labeled with (15)N and (13)C. We describe the automation of these steps and their application to targets chosen from a variety of eukaryotic genomes: Arabidopsis thaliana, human, mouse, rat, and zebrafish. We present protein yields and costs and compare the wheat germ cell-free approach with alternative methods. Finally, we discuss remaining bottlenecks and approaches to their solution.
authors.affiliation: Center for Eukaryotic Structural Genomics, Biochemistry Department, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Dmitriy A
authors.initials: DA
authors.lastname: Vinarov
authors.forename: Carrie L
authors.initials: CL
authors.lastname: Loushin Newman
authors.forename: John L
authors.initials: JL
authors.lastname: Markley
mesh_terms: D000818:Animals; D001331:Automation; D002474:Cell-Free System; D006801:Humans; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D014908:Triticum / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics
chemical_list: D011506:Proteins