TY - JOUR
T1 - Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion
T2 - Case of a nonglycosylated mutant of miraculin, a taste-modifying protein
AU - Ito, Keisuke
AU - Sugawara, Taishi
AU - Koizumi, Ayako
AU - Nakajima, Ken Ichiro
AU - Shimizu-Ibuka, Akiko
AU - Shiroishi, Mitsunori
AU - Asada, Hidetsugu
AU - Yurugi-Kobayashi, Takami
AU - Shimamura, Tatsuro
AU - Asakura, Tomiko
AU - Misaka, Takumi
AU - Iwata, So
AU - Kobayashi, Takuya
AU - Abe, Keiko
N1 - Funding Information:
Acknowledgments This study was supported in part by the ERATO Iwata Human Receptor Crystallography Project (JST) (to S.I.), the Targeted Proteins Research Program (to S.I. and T.M.), a Grant-in-Aid for Scientific Research (B) (20370035) (to T.K.), a Grant-in-Aid for Challenging Exploratory Research (22659059) (to T.K.), a grant from the Research and Development Program for New Bioindustry Initiatives (to K.A.), Grants-in-Aid for Scientific Research (S) (to K.A.), and Grants-in-Aid for JSPS Fellows (to K.I.) in Japan.
PY - 2011/1
Y1 - 2011/1
N2 - 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.
AB - 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.
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U2 - 10.1007/s10529-010-0399-1
DO - 10.1007/s10529-010-0399-1
M3 - Article
C2 - 20936326
AN - SCOPUS:78650515514
VL - 33
SP - 103
EP - 107
JO - Biotechnology Letters
JF - Biotechnology Letters
SN - 0141-5492
IS - 1
ER -