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dc.contributor.authorIto, Keisukeen
dc.contributor.authorSugawara, Taishien
dc.contributor.authorKoizumi, Ayakoen
dc.contributor.authorNakajima, Ken-Ichiroen
dc.contributor.authorShimizu-Ibuka, Akikoen
dc.contributor.authorShiroishi, Mitsunorien
dc.contributor.authorAsada, Hidetsuguen
dc.contributor.authorYurugi-Kobayashi, Takamien
dc.contributor.authorShimamura, Tatsuroen
dc.contributor.authorAsakura, Tomikoen
dc.contributor.authorMasuda, Katsuyoshien
dc.contributor.authorIshiguro, Masajien
dc.contributor.authorMisaka, Takumien
dc.contributor.authorIwata, Soen
dc.contributor.authorKobayashi, Takuyaen
dc.contributor.authorAbe, Keikoen
dc.contributor.alternative小林, 拓也ja
dc.description.abstractBACKGROUND: 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.en
dc.publisherElsevier B.V.en
dc.rights© 2010 Elsevier B.V.en
dc.rightsThis is not the published version. Please cite only the published version.en
dc.subjectSweet proteinen
dc.subjectYeast expression systemen
dc.subjectSteric hindranceen
dc.titleBulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.en
dc.typejournal article-
dc.type.niitypeJournal Article-
dc.identifier.jtitleBiochimica et biophysica actaen
dcterms.accessRightsopen access-
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