Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.

肠激酶是催化水解以调节肠内脏粘膜生物过程的水解酶之一。肠激酶在加速蛋白质消化过程中起着至关重要的作用，因为它通过准确识别和切割特定的肽序列 (Asp)4-Lys 将胰蛋白酶原转化为活性胰蛋白酶。由于其卓越的底物特异性，肠激酶被广泛用作各种生物加工中的多功能分子工具，尤其是从重组蛋白中去除融合标签。尽管具有生物技术重要性，但在细菌中大规模生产可溶性肠激酶仍然是一个未解决的挑战。在这里，我们提出了一种使用由硫氧还蛋白、磷酸甘油酸激酶或麦芽糖结合蛋白组成的串联溶解度增强剂的人肠激酶的有效生产策略。