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The expression of recombinant proteins in microorganism frequently leads to the formation of insoluble aggregates, inclusion bodies (IBs). Thus, the additional in vitro protein refolding process is required to convert inactive IBs into water-soluble active proteins. This study investigated the effect of sulfur residue and hydrophobicity of imidazolium-based room temperature ionic liquids (RTILs) on the refolding of lysozyme as a model protein in the batch dilution method which is the most commonly used refolding method. When lysozyme was refolded in the refolding buffer containing [BF4]-based RTILs with a systematic variety of alkyl chain on cations varying from two to eight, less hydrophobic imidazolium cations having shorter alkyl chains were effective to facilitate lysozyme refolding. Compared to the conventional refolding buffer, 2 times higher lysozyme refolding yield was obtained in 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]) containing refolding buffer. The refolding yield of lysozyme was even more increased by 2.5 times when 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MS]) containing sulfur residue on anion was used. The sulfur residue in [BMIM][MS] is supposed to improve the refolding yield of lysozyme which has 4 intramolecular disulfide bonds. For dilution-based refolding of lysozyme, the optimum concentrations of RTILs in refolding buffer were found to be 1.0 M [EMIM][BF4] and 0.5 M [BMIM][MS], respectively. The optimum temperate for dilution-based refolding of lysozyme with RTILs was 4 °C.
The expression of recombinant proteins in microorganism frequently leads to the formation of insoluble aggregates, inclusion bodies (IBs). Thus, the additional in vitro protein refolding process is required to convert inactive IBs into water-soluble active proteins. This study investigated the effect of sulfur residue and hydrophobicity of imidazolium-based room temperature ionic liquids (RTILs) on the refolding of lysozyme as a model protein in the batch dilution method which is the most commonly used refolding method. When lysozyme was refolded in the refolding buffer containing [BF4] -based RTILs with a systematic variety of alkyl chain on cations varying from two to eight, less hydrophobic imidazolium cations having shorter alkyl chains were effective to facilitate lysozyme refolding. Compared to conventional refolding buffer, 2 times higher lysozyme refolding yield was obtained in 1 -ethyl-3-methylimidazolium tetrafluoroborate ([EMIM] [BF4]) containing refolding buffer. The refol ding yield of lysozyme was even more increased by 2.5 times when 1-butyl-3-methylimidazolium methylsulfate ([BMIM] [MS]) containing sulfur residue on anion was used. The sulfur residue in [BMIM] [MS] is supposed to improve The refolding yield of lysozyme which has 4 intramolecular disulfide bonds. For dilution-based refolding of lysozyme, the optimal concentrations of RTILs in refolding buffer were found to 1.0 M [EMIM] [BF4] and 0.5 M [BMIM] [MS], respectively. The optimum temperate for dilution-based refolding of lysozyme with RTILs was 4 ° C.