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A scalable, GFP-based pipeline for membrane protein overexpression screening and purification

¹ Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden
² MRC Dunn Human Nutrition Unit, Cambridge CB2 2XY, United Kingdom
³ Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA
⁴ Department of Microbiology and Molecular Medicine, Centre Medical, 1211 Geneva, Switzerland

(RECEIVED March 17, 2005; FINAL REVISION April 28, 2005; ACCEPTED April 28, 2005)

We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.

Keywords: Escherichia coli; membrane protein overexpression; membrane protein isolation; membrane protein characterization; GFP

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051466205.

Reprint requests to: Jan-Willem de Gier, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; e-mail: degier{at}dbb.su.se; fax: +46-8-153679.

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