Ultrasensitive Protein Detection Unit, Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Protein Eng Des Sel. 2011 Mar;24(3):261-8. doi: 10.1093/protein/gzq104. Epub 2010 Nov 16.
Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Specifically, protein surface supercharging (N6D, G21D, G22D, N141D, K209E) of the protein increased the solubility more than 100-fold. Replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.
肠肽酶是一种丝氨酸蛋白酶，用于多种生物技术应用。对于许多应用来说，可以使用较小的轻链来避免表达相当大的全酶。重组人肠肽酶轻链（hEPL）表现出高活性，但溶解度和重折叠产率低，目前限制了其在生物技术应用中的使用。在这里，我们描述了几种蛋白质修饰方法，这些方法可以提高人 hEPL 的溶解度和重折叠产率，同时保持酶活性。具体来说，蛋白质表面超电荷（N6D、G21D、G22D、N141D、K209E）使蛋白质的溶解度提高了 100 多倍。用丝氨酸取代游离半胱氨酸残基（C112S）使重折叠产率提高了 50%。这种 C112S 变体的热稳定性也因超电荷而显著提高。本研究表明，即使是温和的蛋白质表面超电荷也会对蛋白质的溶解度和稳定性产生显著影响。
