Hirudin, a blood anticoagulant, is the most potent natural thrombin inhibitor of leech origin. Its application is limited because it is difficult to obtain abundant natural hirudin directly from the leech. Although some bioengineering methods can significantly increase the production of hirudin, the reduced efficacy of recombinant hirudin (rH) remains a critical shortcoming. The lack of sulfation of tyrosine 63 in rH is an important cause of its inadequate performance. This article is the first report of periplasmic co-expression of an rH-I analogue with arylsulfotransferase (ASST) in E. coli BL21(DE3). Co-expressed rH-I analogue with sulfate donor substrate (p-nitrophenyl sulfate potassium) showed anticoagulant (rabbit and goat serum) activity twice more than rH-I analogue expressed without ASST, indicating its potential periplasmic sulfation. Moreover, purified rH-I analogue showed above 4.5 times higher anticoagulant activity compared to therapeutic anti-thrombotic heparin (HE). At the same time, pH-dependent differential solubility was employed to purify rH analogues from fermentation broth, which is a simple, fast and inexpensive purification technology, and can potentially be used for larger scale purification. This will also greatly improve the application of rH in clinical treatment.

水蛭素是一种血液抗凝剂，是水蛭来源的最有效的天然凝血酶抑制剂。由于难以直接从水蛭中获得丰富的天然水蛭素，其应用受到限制。尽管一些生物工程方法可以显着增加水蛭素的产量，但重组水蛭素 (rH) 的功效降低仍然是一个严重的缺点。rH 中酪氨酸 63 缺乏硫酸化是其性能不足的重要原因。本文是关于 rH-I 类似物与芳基磺基转移酶(ASST) 在大肠杆菌BL21(DE3)中周质共表达的首次报道。与硫酸盐供体底物共表达的 rH-I 类似物 ( p-硝基苯硫酸钾）显示出的抗凝（兔和山羊血清）活性比不使用 ASST 表达的 rH-I 类似物高两倍，表明其潜在的周质硫酸化。此外，与治疗性抗血栓形成肝素 (HE) 相比，纯化的 rH-I 类似物显示出高 4.5 倍以上的抗凝活性。同时，利用 pH 依赖性差异溶解度从发酵液中纯化 rH 类似物，这是一种简单、快速且廉价的纯化技术，有可能用于更大规模的纯化。这也将大大提高rH在临床治疗中的应用。