tailieunhanh - Báo cáo khoa học: Wheat germ cell-free platform for eukaryotic protein production

We describe a platform that utilizes wheat germ cell-free technology to pro-duce 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 (25lL) to determine levels of protein expression and solubility. | ễFEBS Journal MINIREVIEW Wheat germ cell-free platform for eukaryotic protein production Dmitriy A. Vinarov Carrie L. Loushin Newman and John L. Markley Center for Eukaryotic StructuralGenomics Biochemistry Department University of Wisconsin-Madison Madison WI USA Keywords cell-free extract in vitro isotopic labeling NMR screening NMR structure determination protein production protein structure transcription translation wheat germ Correspondence J. L. Markley Biochemistry Department University of Wisconsin-Madison 433 Babcock Drive Madison WI 53706 USA Fax 1 608 262 3759 Tel 1 608 263 9349 E-mail markley@ Website http Received 2 May 2006 revised 13 July 2006 accepted 26 July 2006 doi 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 riL to determine levels of protein expression and solubility. The amount of protein produced typically 2-10 Ig 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 15N -labeled amino acids in a 4 mL translation reaction that typically produces 1-3 mg of protein. The 15N -labeled proteins are screened by 1H-15N 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 15N and 13C. We describe the automation of these steps and their application to .