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Title: Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.
Authors: Ito, Keisuke
Sugawara, Taishi
Koizumi, Ayako
Nakajima, Ken-Ichiro
Shimizu-Ibuka, Akiko
Shiroishi, Mitsunori
Asada, Hidetsugu  kyouindb  KAKEN_id  orcid (unconfirmed)
Yurugi-Kobayashi, Takami
Shimamura, Tatsuro  kyouindb  KAKEN_id
Asakura, Tomiko
Masuda, Katsuyoshi
Ishiguro, Masaji
Misaka, Takumi
Iwata, So  kyouindb  KAKEN_id
Kobayashi, Takuya  kyouindb  KAKEN_id
Abe, Keiko
Author's alias: 小林, 拓也
Keywords: Miraculin
Sweet protein
Yeast expression system
Steric hindrance
Issue Date: Sep-2010
Publisher: Elsevier B.V.
Journal title: Biochimica et biophysica acta
Volume: 1800
Issue: 9
Start page: 986
End page: 992
Abstract: 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.
Rights: © 2010 Elsevier B.V.
This is not the published version. Please cite only the published version.
DOI(Published Version): 10.1016/j.bbagen.2010.06.003
PubMed ID: 20542090
Appears in Collections:Journal Articles

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