Optimization of membrane protein production in E. coli and robotic purification

Technology

Protein Expression

Summary

Several membrane proteins have been successfully expressed in E. coli and purified using Maxwell robotic protein purification systems.

Description

The expression of three homologs of Sigma-1 (human, rat, and Guinea pig), OmpG, Estrone sulfatase, AquaporinZ and 2-adrenergic receptor have been optimized by screening for expression in six different E. coli strains (Figure 1A) , and purified by an automated immobilized metal affinity chromatography (IMAC) system (Maxwell 16) [2], and a final clean-up step using HPLC gel filtration. In the case of sigma-1 receptor, the protein was expressed at the small-scale using single colony transformants grown overnight in 0.4 mL of auto-induction medium [1]. The expressed protein was found in the membrane fraction of lysed and sonicated E. coli cells, and was resolubilized in detergent using a high-throughput screen. Finally, sigma-1 was purified using 0.05% fos-choline 12 detergent (FC-12) and IMAC Maxwell system, and polished using a HPLC gel filtration. The Maxwell purification system has also been used for rapid purification of Cell-Free produced bacteriorhodopsin (bR). By combining small-scale E. coli expression strain optimization and robotic automated protein purification using the Maxwell system, we employ relatively simple automated methods for cost-effective isolation of isotopically labeled recombinant membrane proteins at levels sufficient for either functional characterization or structural studies.

Figure


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Figure 1A. Membrane protein E. coli cell line trial C43pRARE2. Figure 1B. Small-scale Maxwell purification of Bacteriorhodopsin. Figure 1C. Sigma 1 receptor detergent solubilization screen with FC-12.

Contact

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

Transmembrane Protein Center

Availability

email for details

Link




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Last edited:Fri 28 Sep 2012 - 4 years, 9 months ago