Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer
Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer
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DOI：
10.1093/jb/mvp184
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发表时间：
2010-03-01
影响因子：
2.7
通讯作者：
Imoto, Taiji
中科院分区：
文献类型：
--
作者：
Kohyama, Keisuke;Matsumoto, Toshihiko;Imoto, Taiji
早些时候，我们正式建立了一种有效的蛋白质再折叠程序，通过梯度去除增溶剂，如尿素[Maeda et al.(1995)]。通过温和去除尿素有效地还原还原溶菌酶。生物工程学报，2011 - 03]。然而，这种方法对不稳定的蛋白质效果较差。本文研究了一种添加蛋白质稳定剂的优良方法，使不稳定蛋白在增溶剂浓度下不形成聚集体的情况下得到合理数量的折叠蛋白。我们检查了许多稳定剂，发现60%的浓缩（2.5 mg/ml）不稳定蛋白可以用40%甘油作为最佳稳定剂进行再折叠。该方法可广泛应用于不稳定蛋白的再折叠。
Earlier, we formally established an effective refolding procedure for a protein by gradient removal of a solubilizer such as urea [Maeda et al. (1995) Effective renaturation of reduced lysozyme by gentle removal of urea. Protein Eng. 8, 201-205]. However, this procedure was less effective for unstable proteins. We developed here an excellent method to add protein stabilizer so as to get reasonable amounts of folded protein under the concentration of solubilizer where the unstable protein does not form aggregate. We examined many stabilizers and found that 60% of a concentrated (2.5 mg/ml) unstable protein can be refolded using 40% glycerol as the best stabilizer. This procedure can be widely applicable for the refolding of unstable proteins.