抄録
Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. Results: As a result, a 2. mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. Conclusions: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. General significance: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.
| 元の言語 | 英語 |
|---|---|
| ページ（範囲） | 986-992 |
| ページ数 | 7 |
| ジャーナル | Biochimica et Biophysica Acta - General Subjects |
| 巻 | 1800 |
| 発行部数 | 9 |
| DOI | |
| 出版物ステータス | 出版済み - 9 1 2010 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Biophysics
- Molecular Biology
- Medicine(all)
これを引用
Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin. / Ito, Keisuke; Sugawara, Taishi; Koizumi, Ayako; Nakajima, Ken ichiro; Shimizu-Ibuka, Akiko; Shiroishi, Mitsunori; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Asakura, Tomiko; Masuda, Katsuyoshi; Ishiguro, Masaji; Misaka, Takumi; Iwata, So; Kobayashi, Takuya; Abe, Keiko.
：: Biochimica et Biophysica Acta - General Subjects, 巻 1800, 番号 9, 01.09.2010, p. 986-992.研究成果: ジャーナルへの寄稿 › 記事
}
TY - JOUR
T1 - Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin
AU - Ito, Keisuke
AU - Sugawara, Taishi
AU - Koizumi, Ayako
AU - Nakajima, Ken ichiro
AU - Shimizu-Ibuka, Akiko
AU - Shiroishi, Mitsunori
AU - Asada, Hidetsugu
AU - Yurugi-Kobayashi, Takami
AU - Shimamura, Tatsuro
AU - Asakura, Tomiko
AU - Masuda, Katsuyoshi
AU - Ishiguro, Masaji
AU - Misaka, Takumi
AU - Iwata, So
AU - Kobayashi, Takuya
AU - Abe, Keiko
PY - 2010/9/1
Y1 - 2010/9/1
N2 - Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. Results: As a result, a 2. mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. Conclusions: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. General significance: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.
AB - Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. Results: As a result, a 2. mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. Conclusions: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. General significance: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.
UR - http://www.scopus.com/inward/record.url?scp=77955281666&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77955281666&partnerID=8YFLogxK
U2 - 10.1016/j.bbagen.2010.06.003
DO - 10.1016/j.bbagen.2010.06.003
M3 - Article
C2 - 20542090
AN - SCOPUS:77955281666
VL - 1800
SP - 986
EP - 992
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0304-4165
IS - 9
ER -