Successful Control of Aggregation and Folding Rates during Refolding of Denatured Lysozyme by Adding N-Methylimidazolium Cations with Various N'-Substituents

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• 作者：Satoshi Yamaguchi ; Etsushi Yamamoto ; Shinya Tsukiji ; Teruyuki Nagamune
• 刊名：Biotechnology Progress
• 出版年：2008
• 出版时间：April 2008
• 年：2008
• 卷：24
• 期：2
• 页码：402 - 408
• 全文大小：106K
• 年卷期：v.24,no.2(April 2008)
• ISSN：1520-6033

文摘

The present study aimed to obtain more effective refolding agents and to understand the influenceof their chemical structures on their function as refolding agents. To achieve these aims, weinvestigated the effects of a large variety of N'-substituted N-methylimidazolium chlorides onthe oxidative refolding of lysozyme in a high throughput manner. Among the molecules examined,N-methylimidazolium cations with a short N'-alkyl chain, such as an N'-ethyl or N'-butyl chain,significantly enhanced the refolding yield compared to conventional refolding additives such asarginine hydrochloride and Triton X-100. Detailed kinetic analyses revealed that the effectivecations selectively decreased the aggregation rate constant (k_A) without any large decreases inthe folding rate constant (k_N). However, when the hydrophobicity of the N'-substituent of thecations was increased, the desirable properties of the short N'-alkyl chain-type cations for proteinrefolding were diminished. Furthermore, increases in the N'-alkyl chain length to an N'-octyl orN'-dodecyl chain drastically decreased the k_A values, thereby increasing the ratio of k_N to k_A,despite the very small k_N values and resulting in enhanced refolding yields. Thus, by tuning thechemical structure of the N'-substituents of N-methylimidazolium chloride, five effective refoldingagents (N'-ethyl-, N'-propyl-, N'-butyl-, N'-pentyl- and N'-isobutyl-N-methylimidazoliumchlorides) were successfully obtained, and the kinetic parameters of folding and aggregationduring the refolding process could be controlled using three different modes.