Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins.
The use of the Protemist XE, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported. The soluble Galdieria sulphuraria protein DCN1 was obtained in greater than 2mg total synthesis yield per mL of reaction mixture from the Protemist XE, and the structure was subsequently solved by X-ray crystallography using material from one 10 mL synthesis (PDB ID: 3KEV). The Protemist XE was also capable of membrane protein translation. Thus human sigma-1 receptor was translated in the presence of unilamellar liposomes and bacteriorhodopsin was translated directly into detergent micelles in the presence of all-trans-retinal. The versatility, ease of use, and compact size of the Protemist XE robot demonstrate its suitability for large-scale synthesis of many classes of proteins.
Beebe
Emily T
ET
Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, 445 Henry Mall, Madison, WI 53706, USA.
Makino
Shin-Ichi
S
Nozawa
Akira
A
Matsubara
Yuko
Y
Frederick
Ronnie O
RO
Primm
John G
JG
Goren
Michael A
MA
Fox
Brian G
BG
D057205:Automation, Laboratory; D002474:Cell-Free System; D003902:Detergents / Q000737:chemistry; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D000461:Rhodophyta / Q000737:chemistry; D012371:Robotics; D014908:Triticum / Q000196:embryology*
D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins