Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: Case of a nonglycosylated mutant of miraculin, a taste-modifying protein

Keisuke Ito, Taishi Sugawara, Ayako Koizumi, Ken Ichiro Nakajima, Akiko Shimizu-Ibuka, Mitsunori Shiroishi, Hidetsugu Asada, Takami Yurugi-Kobayashi, Tatsuro Shimamura, Tomiko Asakura, Takumi Misaka, So Iwata, Takuya Kobayashi, Keiko Abe

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)103-107
Number of pages5
JournalBiotechnology letters
Volume33
Issue number1
DOIs
Publication statusPublished - Jan 1 2011

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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    Ito, K., Sugawara, T., Koizumi, A., Nakajima, K. I., Shimizu-Ibuka, A., Shiroishi, M., Asada, H., Yurugi-Kobayashi, T., Shimamura, T., Asakura, T., Misaka, T., Iwata, S., Kobayashi, T., & Abe, K. (2011). Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: Case of a nonglycosylated mutant of miraculin, a taste-modifying protein. Biotechnology letters, 33(1), 103-107. https://doi.org/10.1007/s10529-010-0399-1