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Functional expression of eukaryotic membrane proteins in Lactococcus lactis

The Medical Research Council, Dunn Human Nutrition Unit, Cambridge CB2 2XY, United Kingdom

(RECEIVED July 8, 2005; FINAL REVISION September 16, 2005; ACCEPTED September 16, 2005)

The overproduction of eukaryotic membrane proteins is a major impediment in their structural and functional characterization. Here we have used the nisin-inducible expression system of Lactococcus lactis for the overproduction of 11 mitochondrial transport proteins from yeast. They were expressed at high levels in a functional state in the cytoplasmic membrane. The results also show that the level of expression is influenced by the N-terminal regions of the transporters. Expression levels were improved >10-fold either by replacing or truncating these regions or by adding lactococcal signal peptides. The observed expression levels are now compatible with a realistic exploration of crystallization conditions. The lactococcal expression system may be used for the high-throughput functional characterization of eukaryotic membrane proteins and structural genomics.

Keywords: eukaryotic membrane proteins; mitochondrial carriers; nisin-inducible expression system; Lactococcus lactis; transport

Abbreviations: NMR. nuclear magnetic resonance • TOM, translo-case of the outer membrane • TIM, translocase of the inner membrane.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051689905.

Reprint requests to: Edmund R.S. Kunji, The Medical Research Council, Dunn Human Nutrition Unit, Hills Road, Wellcome Trust/MRC Building, Cambridge CB2 2XY, UK; e-mail: ek{at}mrc-dunn.cam.ac.uk; fax: +44-1223-252875.

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