Daugherty D L, Rozema D, Hanson P E, Gellman S H
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 1998 Dec 18;273(51):33961-71. doi: 10.1074/jbc.273.51.33961.
The power of genetic engineering methods, along with increasing genomic information, makes heterologous expression of proteins an extremely important biochemical tool. Unfortunately, proteins obtained in this way often are not in their native form, and folding becomes a crucial step in protein production. We have recently developed a strategy that promotes the folding of chemically denatured proteins via the sequential addition of low molecular weight "artificial chaperones." Here we describe in detail the application of this method to porcine heart citrate synthase. Refolding yields of as high as 65% have been achieved. Mechanistic studies indicate that there are significant differences between artificial chaperone-assisted refolding of citrate synthase and artificial chaperone-assisted refolding of two other proteins that have been examined, carbonic anhydrase B (Rozema, D., and Gellman, S. H. (1996) J. Biol. Chem. 271, 3478-3487) and lysozyme (Rozema, D., and Gellman, S. H. (1996) Biochemistry 35, 15760-15771). The differences among these three test proteins reveal the range of procedural variation that must be considered in the application of the artificial chaperone method to new proteins.
基因工程方法的强大功能，再加上日益增多的基因组信息，使得蛋白质的异源表达成为一种极其重要的生化工具。不幸的是，通过这种方式获得的蛋白质往往并非天然形式，而折叠成为蛋白质生产中的关键步骤。我们最近开发了一种策略，通过依次添加低分子量的“人工伴侣分子”来促进化学变性蛋白质的折叠。在此，我们详细描述该方法在猪心脏柠檬酸合酶上的应用。已实现高达65%的复性产率。机理研究表明，柠檬酸合酶的人工伴侣辅助复性与另外两种已研究蛋白质（碳酸酐酶B（Rozema, D., and Gellman, S. H. (1996) J. Biol. Chem. 271, 3478 - 3487）和溶菌酶（Rozema, D., and Gellman, S. H. (1996) Biochemistry 35, 15760 - 15771））的人工伴侣辅助复性之间存在显著差异。这三种测试蛋白质之间的差异揭示了在将人工伴侣方法应用于新蛋白质时必须考虑的程序变化范围。
