Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant.
Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.
Université Grenoble 1, Institut de Biologie Structurale, Grenoble Cedex 1, France.
Iulia
I
Blesneac
Stéphanie
S
Ravaud
Céline
C
Juillan-Binard
Laure-Anne
LA
Barret
Manuela
M
Zoonens
Ange
A
Polidori
Bruno
B
Miroux
Bernard
B
Pucci
Eva
E
Pebay-Peyroula
D000818:Animals; D002417:Cattle; D002474:Cell-Free System / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D015870:Gene Expression; D006845:Hydrocarbons, Fluorinated / Q000737:chemistry*; D007473:Ion Channels / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013501:Surface-Active Agents / Q000737:chemistry*; D000071256:Uncoupling Protein 1
D006845:Hydrocarbons, Fluorinated; D007473:Ion Channels; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D013501:Surface-Active Agents; C000604373:Ucp1 protein, rat; D000071256:Uncoupling Protein 1