Purification and characterization of the creatine transporter expressed at high levels in HEK293 cells.
The bovine creatine transporter (CreaT) has been purified from membranes of HEK293 cells stably expressing high levels of the transporter. Membranes were solubilized with decyl maltoside and the CreaT was purified (90% pure) by affinity chromatography on wheat germ agglutinin (WGA)-Sepharose and gel-filtration. The CreaT was shown to be an approximately 70 kDa glycoprotein by SDS-polyacrylamide gel electrophoresis and Western blotting. Identification of the CreaT was confirmed by sequencing tryptic peptides by mass spectrometry. Laser light scattering showed the majority of the CreaT to be present as a 224 kDa species. Additional purification was obtained when the Creat was eluted from the WGA column and purified by gel-filtration in Fos-choline 12 instead of decyl maltoside, followed by a second WGA affinity step to exchange the detergent for sodium cholate. This resulted in a 30-fold purification (95% purity) of the approximately 70kDa CreaT, with a yield of 15%. From this, it is estimated that the CreaT comprises approximately 3% of total HEK293-CreaT membrane protein. Gel-filtration showed the transporter to migrate with an apparent molecular mass of 210 kDa. Circular dichroism showed a predominantly alpha-helical structure, consistent with the 12 transmembrane domains predicted for the transporter. This work has enabled the purification of the CreaT in amounts ( approximately 100 microg) that make it feasible to consider structural studies of a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter family.
West
Mark
M
Biochemistry and Cell Biology Group, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Park
Daniel
D
Dodd
Joanna R
JR
Kistler
Joerg
J
Christie
David L
DL
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D008970:Molecular Weight
D026901:Membrane Transport Proteins; C080679:creatine transporter