Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae.
Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 mu plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.
Sawamiphak
Suphansa
S
Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Phayathai, Rajathevee Bangkok 10400, Thailand.
Sophasan
Samaisukh
S
Endou
Hitoshi
H
Boonchird
Chuenchit
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003062:Codon; D006031:Glycosylation; D008969:Molecular Sequence Data; D027362:Organic Anion Transport Protein 1 / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D013696:Temperature; D010130:p-Aminohippuric Acid / Q000378:metabolism
D003062:Codon; D027362:Organic Anion Transport Protein 1; D011994:Recombinant Proteins; C421043:Slc22a6 protein, rat; D010130:p-Aminohippuric Acid