Cell-free protein synthesis technology in NMR high-throughput structure determination.
This chapter describes the current implementation of the cell-free translation platform developed at the Center for Eukaryotic Structural Genomics (CESG) and practical aspects of the production of stable isotope-labeled eukaryotic proteins for NMR structure determination. Protocols are reported for the use of wheat germ cell-free translation in small-scale screening for the level of total protein expression, the solubility of the expressed protein, and the success in purification as predictive indicators of the likelihood that a protein may be obtained in sufficient quantity and quality to initiate structural studies. In most circumstances, the small-scale reactions also produce sufficient protein to permit bioanalytical and functional characterizations. The protocols incorporate the use of robots specialized for small-scale cell-free translation, large-scale protein production, and automated purification of soluble, His(6)-tagged proteins. The integration of isotopically labeled proteins into the sequence of experiments required for NMR structure determination is outlined, and additional protocols for production of integral membrane proteins in the presence of either detergents or unilamellar liposomes are presented.
Makino
Shin-ichi
S
Department of Biochemistry, Center for Eukaryotic Structural Genomics, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI, USA.
Goren
Michael A
MA
Fox
Brian G
BG
Markley
John L
JL
D002474:Cell-Free System; D057166:High-Throughput Screening Assays; D006639:Histidine / Q000378:metabolism; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012639:Seeds / Q000378:metabolism; D014908:Triticum / Q000196:embryology / Q000235:genetics / Q000378:metabolism*
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine