Premium
Wheat germ cell‐free platform for eukaryotic protein production
Author(s) -
Vinarov Dmitriy A.,
Newman Carrie L. Loushin,
Markley John L.
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05434.x
Subject(s) - protein biosynthesis , wheat germ , protein purification , cell free protein synthesis , biology , biochemistry , polyacrylamide gel electrophoresis , solubility , dna , amino acid , germ , chemistry , enzyme , microbiology and biotechnology , organic chemistry
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 µL) to determine levels of protein expression and solubility. The amount of protein produced (typically 2–10 µg) 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.