Kim Young Su, Lee Hye-Jeong, Park Sang-Hyun, Kim Yeu-Chun, Ahn Jungoh
Department of Chemical and Biomolecular Engineering, KAIST, Daejeon 34141, Republic of Korea.
Biotechnology Process Engineering Center, KRIBB, Cheongju 28116, Republic of Korea.
Biotechnol Rep (Amst). 2021 May 5;30:e00626. doi: 10.1016/j.btre.2021.e00626. eCollection 2021 Jun.
Human enterokinase light chain (hEK) specifically cleaves the sequence (Asp)-Lys↓X (DK), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEK production from is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEK from by expressing the hEK variant C112S fused with maltose-binding protein (MBP) through DK and molecular chaperons including GroEL/ES. The fusion protein self-cleaved thereby removing the MBP in the cells. Thus, the self-cleaved hEK variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEK variant exhibiting an enzymatic activity of 3.1 × 10 U/mL (9.934 × 10 U/mg). The approaches presented here greatly simplify the purification of hEK from without requiring refolding processes.
人肠激酶轻链（hEK）特异性切割序列（天冬氨酸）-赖氨酸↓X（DK），使其成为重组融合蛋白位点特异性切割常用的酶。然而，由于分子内二硫键的存在，从大肠杆菌中生产hEK受到限制。在此，我们提出了通过表达与麦芽糖结合蛋白（MBP）融合的hEK变体C112S，并通过DK和包括GroEL/ES在内的分子伴侣从大肠杆菌中获得可溶性和活性hEK的策略。融合蛋白在大肠杆菌细胞中自切割，从而去除MBP。因此，自切割的hEK变体被释放到培养基中。使用HisTrap™ 色谱进行一步纯化，纯化后的hEK变体显示出3.1×10 U/mL（9.934×10 U/mg）的酶活性。本文提出的方法极大地简化了从大肠杆菌中纯化hEK的过程，无需重折叠过程。
