CPN spol. s r.o., Dolní Dobrouč 401, 56102 Dolní Dobrouč, Czech Republic.
J Biotechnol. 2011 Oct 20;156(1):67-75. doi: 10.1016/j.jbiotec.2011.08.017. Epub 2011 Aug 23.
Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.
人肠激酶（肠肽酶，rhEP）是一种在小肠近端表达的丝氨酸蛋白酶，通过内切酶切割将无活性的胰蛋白酶原转化为有活性的胰蛋白酶。靶位点的高度特异性使肠激酶成为在定义的切割位点切割融合蛋白的理想工具。成熟的活性酶由两条二硫键连接的多肽链组成。重链将酶锚定在肠刷状缘膜上，而轻链代表催化酶亚基。编码人肠激酶轻链的合成基因与 C 末端的 His 标签融合，以利于蛋白纯化，该基因被克隆到毕赤酵母表达质粒中，受诱导型 AOX1 或组成型启动子 GAP 和 AAC 的控制。优化了培养介质和条件，以及目标蛋白的分离和纯化。在摇瓶实验中获得了高达 4mg/L 的 rhEP，当在实验室规模的发酵罐中培养时，表达水平达到约 60-70mg/L。组成型表达株比诱导型表达株更有效率，所需劳动力更少。rhEP 被固定在 AV 100 吸附剂（Iontosorb）上，以允许肠激酶重复使用，显示出湿基质 4U/mL 的比活性。
