Authors: Keisuke Ito Taishi Sugawara Ayako Koizumi Kenichiro Nakajima Akiko ShimizuIbuka Mitsunori Shiroishi Hidetsugu Asada Takami YurugiKobayashi Tatsuro Shimamura Tomiko Asakura Takumi Misaka So Iwata Takuya Kobayashi Keiko Abe
Publish Date: 2010/10/09
Volume: 33, Issue: 1, Pages: 103-107
Abstract
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 systemIntentional mutagenesis studies should consider the size of the library and the time required for expression screening Here we proposed a cysteinetoserine shuffling mutation strategy CS shuffling using a Saccharomyces cerevisiae expression system This strategy of sitedirected shuffling mutagenesis of cysteinetoserine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression In the case of a nonglycosylated mutant of the tastemodifying 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 expressionThis study was supported in part by the ERATO Iwata Human Receptor Crystallography Project JST to SI the Targeted Proteins Research Program to SI and TM a GrantinAid for Scientific Research B 20370035 to TK a GrantinAid for Challenging Exploratory Research 22659059 to TK a grant from the Research and Development Program for New Bioindustry Initiatives to KA GrantsinAid for Scientific Research S to KA and GrantsinAid for JSPS Fellows to KI in Japan
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