Biotechnology Letters

, Volume 33, Issue 1, pp 103–107 | Cite as

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
Original Research Paper

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.

Keywords

Protein secretion Mutagenesis Screening method Disulfide bond Cysteine Yeast expression system 

References

  1. Hagihara Y, Kim PS (2002) Toward development of a screen to identify randomly encoded, foldable sequences. Proc Natl Acad Sci USA 99:6619–6624CrossRefPubMedGoogle Scholar
  2. Ito K et al (2007) Microbial production of sensory-active miraculin. Biochem Biophys Res Commun 360:407–411CrossRefPubMedGoogle Scholar
  3. Ito K et al (2008) Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae. Biochem Biophys Res Commun 371:841–845CrossRefPubMedGoogle Scholar
  4. Kawamura S et al (2008) Role of disulfide bonds in goose-type lysozyme. FEBS J 275:2818–2830CrossRefPubMedGoogle Scholar
  5. Koschorreck K et al (2009) Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis. BMC Biotechnol 9:12CrossRefPubMedGoogle Scholar
  6. Kurihara K, Beidler LM (1969) Mechanism of the action of taste-modifying protein. Nature 222:1176–1179CrossRefPubMedGoogle Scholar
  7. Liu Y et al (2005) Increase of soluble expression in Escherichia coli cytoplasm by a protein disulfide isomerase gene fusion system. Protein Expr Purif 44:155–161CrossRefPubMedGoogle Scholar
  8. Newstead S et al (2007) High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 104:13936–13941CrossRefPubMedGoogle Scholar
  9. Sakoh-Nakatogawa M et al (2009) Roles of protein-disulfide isomerase-mediated disulfide bond formation of yeast Mnl1p in endoplasmic reticulum-associated degradation. J Biol Chem 284:11815–11825CrossRefPubMedGoogle Scholar
  10. Sugawara T et al (2009) Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae. Biochem Biophys Res Commun 382:704–710CrossRefPubMedGoogle Scholar
  11. Uchiyama H et al (2000) Directed evolution to improve the thermostability of prolyl endopeptidase. J Biochem 128:441–447PubMedGoogle Scholar
  12. Yuan S et al (2004) The role of thioredoxin and disulfide isomerase in the expression of the snake venom thrombin-like enzyme calobin in Escherichia coli BL21 (DE3). Protein Expr Purif 38:51–60CrossRefPubMedGoogle Scholar
  13. Zamyatnin AA (1972) Protein volume in solution. Prog Biophys Mol Biol 24:107–123CrossRefPubMedGoogle Scholar
  14. Zavodszky M et al (2001) Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V. Protein Sci 10:149–160CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Keisuke Ito
    • 1
    • 2
  • Taishi Sugawara
    • 1
  • Ayako Koizumi
    • 1
  • Ken-ichiro Nakajima
    • 1
  • Akiko Shimizu-Ibuka
    • 1
  • Mitsunori Shiroishi
    • 3
  • Hidetsugu Asada
    • 3
  • Takami Yurugi-Kobayashi
    • 3
  • Tatsuro Shimamura
    • 3
  • Tomiko Asakura
    • 1
  • Takumi Misaka
    • 1
  • So Iwata
    • 3
    • 4
    • 5
  • Takuya Kobayashi
    • 3
    • 4
    • 6
  • Keiko Abe
    • 1
    • 7
  1. 1.Department of Applied Biological Chemistry, Graduate School of Agricultural and Life SciencesThe University of TokyoTokyoJapan
  2. 2.Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental SciencesUniversity of ShizuokaShizuokaJapan
  3. 3.Iwata Human Receptor Crystallography ProjectERATO, JSTKyotoJapan
  4. 4.Department of Medical Chemistry, Faculty of MedicineKyoto UniversityKyotoJapan
  5. 5.Division of Molecular Biosciences, Membrane Protein Crystallography GroupImperial College LondonLondonUK
  6. 6.Laboratory of Cell Biology, 3rd Flr., Bldg. A, Graduate School of MedicineKyoto UniversityKyotoJapan
  7. 7.Laboratory of Biological Function, 3rd Flr., Bldg. 7B, Graduate School of Agricultural and Life SciencesThe University of TokyoTokyoJapan

Personalised recommendations