title: Functional expression of eukaryotic membrane proteins in Lactococcus lactis.
abstract: 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.
authors.affiliation: The Medical Research Council, Dunn Human Nutrition Unit, Hills Road, Wellcome Trust/MRC Building, Cambridge CB2 2XY, UK.
authors.forename: Magnus
authors.initials: M
authors.lastname: Monné
authors.forename: Ka Wai
authors.initials: KW
authors.lastname: Chan
authors.forename: Dirk-Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.forename: Edmund R S
authors.initials: ER
authors.lastname: Kunji
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D013294:Lactococcus lactis / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins