Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins

Technology

Protein Expression

Summary

The use of the Protemist XE, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported.

Description

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.

Figure


uploads/Protemist_XE.jpg

Figure 1. The Protemist XE, a cell-free translation robot that uses a discontinuous-batch method. The feed buffer reservoir (A) and mRNA reservoir (B) are used to refill the reaction chamber (C) after 50% reduction in volume by ultrafiltration using a VIVFLOW 50 tangential flow ultrafiltration cassette (D). A stand-alone PC is used to control the pumping system.

Publication

Beebe ET, Makino S, Nozawa A, Matsubara Y, Frederick RO, Primm JG, Goren MA, Fox BG. Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins. N Biotechnol 28(3):239-49 (2011). PubMed ID: 20637905 | Search SBKB Publications portal | PMC Link

Contact

Brian G. Fox (bgfox@biochem.wisc.edu)

Transmembrane Protein Center

Link




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Last edited:Tue 08 Jan 2013 - 4 years, 5 months ago