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タイトル: | Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein. |
著者: | Ito, Keisuke Sugawara, Taishi Koizumi, Ayako Nakajima, Ken-ichiro Shimizu-Ibuka, Akiko Shiroishi, Mitsunori Asada, Hidetsugu https://orcid.org/0000-0001-6255-4728 (unconfirmed) Yurugi-Kobayashi, Takami Shimamura, Tatsuro Asakura, Tomiko Misaka, Takumi Iwata, So Kobayashi, Takuya Abe, Keiko |
著者名の別形: | 小林, 拓也 |
キーワード: | Protein secretion Mutagenesis Screening method Disulfide bond Cysteine Yeast expression system |
発行日: | Jan-2011 |
出版者: | Springer Science+Business Media B.V. |
誌名: | Biotechnology letters |
巻: | 33 |
号: | 1 |
開始ページ: | 103 |
終了ページ: | 107 |
抄録: | 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. |
著作権等: | The final publication is available at www.springerlink.com This is not the published version. Please cite only the published version. 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。 |
URI: | http://hdl.handle.net/2433/134585 |
DOI(出版社版): | 10.1007/s10529-010-0399-1 |
PubMed ID: | 20936326 |
出現コレクション: | 学術雑誌掲載論文等 |
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