SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.
The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.
Sagné
C
C
CNRS URA 1112, Institut de Biologie Physico-Chimique, Paris, France.
Isambert
M F
MF
Henry
J P
JP
Gasnier
B
B
D000818:Animals; D001345:Autoradiography; D001692:Biological Transport; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008961:Models, Structural; D008970:Molecular Weight; D009479:Neuropeptides; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D012967:Sodium Dodecyl Sulfate