Protein refolding at high concentration using size‐exclusion chromatography
Protein refolding at high concentration using size‐exclusion chromatography
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DOI：
10.1002/(sici)1097-0290(19960405)50:1
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发表时间：
1996-04
影响因子：
3.8
通讯作者：
B. Batas;J. Chaudhuri
中科院分区：
文献类型：
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作者：
B. Batas;J. Chaudhuri
已经开发出一种在单次操作中提高重折叠产量和增加重折叠蛋白质浓度的新方法。该方法使用尺寸排阻色谱基质来执行缓冲液交换、聚集体去除和折叠反应。凝胶过滤介质中蛋白质扩散的减少已被证明可以抑制部分折叠分子的非特异性相互作用，从而减少聚集。使用Sephacryl S-100 (HR) 成功地将鸡蛋清溶菌酶 (HEWL) 和牛碳酸酐酶 (CAB) 从初始蛋白质浓度高达 80 mg/mL 重折叠。溶菌酶的聚集反应减少，并且仅在使用的最高蛋白质浓度下检测到。溶菌酶的平均回收率为63%，平均比活力为104%。碳酸酐酶实验还表明，聚集被抑制，从柱中平均蛋白质回收率为 56%，比活性为 81%。该过程可以一步实现活性物质的重折叠和纯化。 © 1996 约翰威利父子公司。
A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size‐exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel‐filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S‐100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. © 1996 John Wiley & Sons, Inc.