Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria.
J Chromatogr A. 2010 Sep 17;1217(38):5950-6. doi: 10.1016/j.chroma.2010.07.053. Epub 2010 Jul 22.
Matrix-assisted refolding is an excellent technique for performing refolding of recombinant proteins at high concentration because aggregation during refolding is partially suppressed. The autoprotease N(pro) and its engineered mutant EDDIE can be efficiently refolded on cation-exchangers. In the current work, denatured fusion proteins were loaded at different column saturations (5 and 50 mg mL(-1) gel), and refolding and self-cleavage were initiated during elution. The contact time of the protein with the matrix significantly influenced the refolding rate and yield. On POROS 50 HS, the refolding rate was comparable to a batch refolding process, but yield was substantially higher; at a protein concentration of 1.55 mg mL(-1), an almost complete conversion was observed. With Capto S, the rate of self-cleavage increased by a factor of 20 while yield was slightly reduced. Processing the autoprotease fusion protein on Capto S at a high protein loading of 50 mg mL(-1) gel and short contact time (0.5h) yielded the highest productivity.
基质辅助复性是一种在高浓度下进行重组蛋白复性的极好技术，因为复性过程中的聚集部分受到抑制。自蛋白酶 N(pro)及其工程突变体 EDDIE 可以在阳离子交换剂上有效地进行复性。在当前的工作中，变性融合蛋白在不同的柱饱和度（5 和 50mg/ml 凝胶）下加载，并在洗脱过程中启动复性和自我切割。蛋白质与基质的接触时间显著影响复性速率和收率。在 POROS 50HS 上，复性速率与批量复性过程相当，但收率显著提高；在蛋白质浓度为 1.55mg/ml 时，几乎完全转化。在 Capto S 上，自我切割的速率提高了 20 倍，而收率略有降低。在高蛋白质装载量为 50mg/ml 凝胶和短接触时间（0.5h）的情况下，在 Capto S 上处理自蛋白酶融合蛋白可获得最高的生产率。
