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Engineering membrane protein overproduction in Escherichia coli.

¹ Department of Biochemistry and Biophysics, Stockholm University, S-106 91 Stockholm, Sweden
² Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-117 38 Stockholm, Sweden

(RECEIVED September 17, 2007; FINAL REVISION December 20, 2007; ACCEPTED December 21, 2007)

Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold.

Keywords: random mutagenesis; screening; membrane protein; overexpression; colony filtration; in vitro evolution

Reprint requests to: Pär Nordlund, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-117 38 Stockholm, Sweden; e-mail: par.nordlund{at}ki.se; fax: 46-8-524-868-50.

Abbreviations: CoFi, colony filtration; IMP, integral membrane protein.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073242508.

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