Expression and purification of bacteriorhodopsin using Protemist XE with detergents

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 2 mg 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.

We demonstrated the efficacy of the Protemist XE (Panel A), a compact cell-free translation system capable of producing yields of membrane protein sufficient for NMR or crystallography. This tangential-flow dialysis system was used with optimized wheat germ extract and buffer conditions to synthesize the 7-TM Halobacter proton pump bacteriorhodopsin (bR) in the presence of 0.2% CHAPS and 0.1 mM all-trans-retinal (Panel B). The initial synthesized yield was 5 mg of selenomethionine-labeled bR precipitate from a 10 mL reaction.

Further adjustments of detergent conditions in both the reaction and feeding mixtures are expected to improve these yields. Greater than 95% bR was solubilized in 0.5% FC-12. This material was purified by IMAC and buffer exchanged into 0.05% DDM. The protein was gel-filtered in 5 mM MES, pH 5.5, 0.025% DDM, 100 mM NaCl, and 0.3 mM TCEP, producing a peak corresponding to a protein:detergent complex size of 135 kDa (Panel C), consistent with dimeric or trimeric bR in DDM micelles. 1.3 mg of deeply pigmented protein was purified with an optical purity (A280/A555) of 2.2 (Panels D and E). In comparison, the A280/A565 ratio of Halobacter bR was 1.9. The protein was stable for weeks at 4°C in these buffer conditions.

Circular Dichroism spectroscopy showed that the cell-free bR was strongly alpha-helical and spectrally equivalent to bR from Halobacter membranes (Panel F). Peak flattening evident in the Halobacter bR preparation was likely due to the presence of lipids, which were lacking in the detergent-synthesized cell-free preparation. Crystallization trials are underway for the cell-free bacteriorhodopsin using hanging drop, bicelle, and lipidic cubic phase methods.

Figure


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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." Nat. Biotechnol. 28:239-49 (2011). PubMed ID: 20637905 | Search SBKB Publications portal | PMC Link

Contact

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

Transmembrane Protein Center

Availability

email for details

Link




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Last edited:Thu 27 Sep 2012 - 4 years, 9 months ago