Addition of signal leader sequences to the N-termini of olfactory receptor proteins enhances their expression in Xenopus oocytet.
Several recent papers have reported the difficulties in expressing olfactory receptor proteins (ORs) in heterologous systems, and proposed that some sequences in ORs have negative effects on their efficient expression. To obtain an efficient expression system of ORs, we modified N-terminal sequences of ORs through the addition of exogenous sequences. Three kinds of sequences, designated as 5HT, V, and VL, were used. 5HT and V corresponded to the signal leader (SL) sequences of 5HT 3R and VIPR, respectively. VL corresponded to the first extracellular region of VIPR containing the SL sequence and three potential asparagine- (Asn-) linked glycosylation sites. The myc epitope was also added to the C-termini of the sequences. Several ORs including 17 of rat, GUST43 of rat, Y1 of medaka, FOR1-3 of pufferfish, 47E of carp, and ODR-10 of nematode were subjected to the modifications, and the RNAs encoding modified ORs were injected into Xenopus oocytes. The membrane fraction of the oocytes were analyzed by Western blotting to examine the expression of the proteins. In the cases of ORs modified with 5HT and V, only ODR-10 and 47E, both of which have more than two Asn-linked glycosylation sites in their extracellular regions, were detected as the bands of predicted molecular weights. On the other hand, most of the ORs modified with VL showed the bands of predicted molecular weights. These results suggest that SL sequences together with potential Asn-linked glycosylation sites have positive effects on the expression of ORs in heterologous systems.
Yasuoka
A
A
Bio-oriented Technology Research Advancement Institution, Oomiya, Saitama, Japan. ayas@mail.ecc.u-tokyo.ac.jp
Emori
Y
Y
Abe
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D002347:Carps; D002397:Catfishes; D005399:Fishes; D006031:Glycosylation; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D009990:Oryzias; D011487:Protein Conformation; D011489:Protein Denaturation; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D018035:Receptors, Odorant / Q000096:biosynthesis* / Q000235:genetics; D013329:Structure-Activity Relationship; D014981:Xenopus
D021382:Protein Sorting Signals; D018035:Receptors, Odorant; D001216:Asparagine