Article

Genetic selection system for improving recombinant membrane protein expression in E. coli

Elizabeth Massey-Gendel 1, Anni Zhao 1, Gabriella Boulting 1 a, Hye-Yeon Kim 2 b, Michael A. Balamotis 3, Len M. Seligman 4, Robert K. Nakamoto 5, James U. Bowie 1 2 *

1Department of Chemistry and Biochemistry, University of California, Los Angeles, California 2UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, California 3Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 4Department of Biology, Pomona College, Claremont, California 5Department of Molecular Physiology & Biological Physics, University of Virginia, Charlottesville, Virginia

email: James U. Bowie (bowie@mbi.ucla.edu)

^*Correspondence to James U. Bowie, Boyer Hall, University of California, Los Angeles, 611 Charles E. Young Dr. E, Los Angeles, CA 90095-1570

^aCurrent address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA

^bCurrent address: Center for Magnetic Resonance Research, Korea Basic Science Institute, 113 Gwahangno, Yusung-gu, Daejeon, 305-333, Korea

Funded by:
 NIH Roadmap Grant; Grant Number: RO1 GM075922
 NIH/NIGMS Ruth L. Kirschstein National Research Service Award; Grant Number: GM07185

Keywords

recombinant membrane protein expression • E. coli • plasmid curing

Abstract

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.

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Received: 17 September 2008; Revised: 11 November 2008; Accepted: 12 November 2008

Digital Object Identifier (DOI)

10.1002/pro.39  About DOI