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Title: Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein.
Authors: Ito, Keisuke
Sugawara, Taishi
Koizumi, Ayako
Nakajima, Ken-ichiro
Shimizu-Ibuka, Akiko
Shiroishi, Mitsunori
Asada, Hidetsugu  kyouindb  KAKEN_id  orcid https://orcid.org/0000-0001-6255-4728 (unconfirmed)
Yurugi-Kobayashi, Takami
Shimamura, Tatsuro  kyouindb  KAKEN_id
Asakura, Tomiko
Misaka, Takumi
Iwata, So  kyouindb  KAKEN_id
Kobayashi, Takuya  kyouindb  KAKEN_id
Abe, Keiko
Author's alias: 小林, 拓也
Keywords: Protein secretion
Mutagenesis
Screening method
Disulfide bond
Cysteine
Yeast expression system
Issue Date: Jan-2011
Publisher: Springer Science+Business Media B.V.
Journal title: Biotechnology letters
Volume: 33
Issue: 1
Start page: 103
End page: 107
Abstract: PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.
Rights: The final publication is available at www.springerlink.com
この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。This is not the published version. Please cite only the published version.
URI: http://hdl.handle.net/2433/134585
DOI(Published Version): 10.1007/s10529-010-0399-1
PubMed ID: 20936326
Appears in Collections:Journal Articles

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