Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin.
The membrane protein prestin is the voltage-sensitive molecular motor underlying somatic electromotility of outer hair cells. In order to produce adequate quantities to perform structural and functional studies, we cloned and expressed in bacterial systems three variants of the cytosolic C-terminal STAS domain of prestin from Rattus norvegicus. While the expression level of the longer form of the C-terminal domain (fragment [505-744]) was very low or absent, we succeeded in the overexpression of two shorter fragment of the STAS domain (fragments [529-744], PreCD(L), and [529-720], PreCD(S)). These two polypeptides were purified to homogeneity and characterised by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The two proteins possess a three-dimensional structure and show a great tendency to aggregate. In particular, PreCD(L) is present in solution mainly as dimers and tetramers. These data correlate with that of full-length prestin that forms stable tetramers, suggesting that the C-terminal domain play an important role in modulating the properties of the entire prestin.
Pasqualetto
Elisa
E
Department of Chemical Sciences, University of Padua, via Marzolo 1, 35131 Padua, Italy.
Seydel
Anke
A
Pellini
Alberto
A
Battistutta
Roberto
R
D027321:Anion Transport Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D017920:Antiporters / Q000096:biosynthesis* / Q000302:isolation & purification; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D000076988:Sulfate Transporters
D027321:Anion Transport Proteins; D017920:Antiporters; D011506:Proteins; D011994:Recombinant Proteins; C056459:Slc26a1 protein, rat; C409207:Slc26a5 protein, rat; D000076988:Sulfate Transporters