Yazdanparast Razieh, Khodarahmi Reza
Institute of Biochemistry and Biophysics, PO Box 13145-1384, The University of Tehran, Tehran, Iran.
Int J Biol Macromol. 2007 Mar 10;40(4):319-26. doi: 10.1016/j.ijbiomac.2006.08.018. Epub 2006 Sep 12.
Insoluble beta-cyclodextrin (beta-CD) copolymers have been used for the refolding of thermally and/or chemically denatured carbonic anhydrase with refolding yield of 40% using 300 mg of the copolymer/ml refolding solution containing 0.042 mg/ml protein. In an attempt to enhance the refolding yield with lower quantities of the copolymer, a new beta-CD-rich copolymer with higher beta-CD content was synthesized. Regarding the need for rapid stripping of the detergent molecules from the detergent-protein complexes formed in the capture step of the technique (artificial chaperone-assisted refolding), experimental variables (e.g. copolymer and the protein contents) were optimized to improve the refolding yields along with depressing the aggregate formation. In addition, comparative studies using different ionic detergents and the copolymer were conducted to get a more comprehensive understanding of the detergent's tail length in the stripping step of the process. Our results indicated that under the optimal developed refolding environment, the denatured CA was refolded with a yield of 75% using only 5mg of the copolymer/1.2 ml refolding solution containing 0.0286 mg/ml protein. Taking into account the recycling potential of the copolymer, the new resin, with significant cost-cutting capability, is a suitable candidate for industrial applications.
不溶性β-环糊精（β-CD）共聚物已用于热变性和/或化学变性碳酸酐酶的复性，在含有0.042 mg/ml蛋白质的复性溶液中使用300 mg共聚物/ml时，复性产率为40%。为了用较少量的共聚物提高复性产率，合成了一种β-CD含量更高的新型富含β-CD的共聚物。考虑到在该技术（人工伴侣辅助复性）的捕获步骤中形成的洗涤剂-蛋白质复合物需要快速去除洗涤剂分子，对实验变量（如共聚物和蛋白质含量）进行了优化，以提高复性产率并抑制聚集体形成。此外，还进行了使用不同离子洗涤剂和共聚物的比较研究，以更全面地了解该过程中去除步骤中洗涤剂的尾长。我们的结果表明，在优化的复性环境下，仅使用5mg共聚物/1.2 ml含有0.0286 mg/ml蛋白质的复性溶液，变性的CA复性产率可达75%。考虑到共聚物的循环利用潜力，这种具有显著成本降低能力的新型树脂是工业应用的合适候选材料。
