Expression and purification of soluble and active human enterokinase light chain in Escherichia coli

Young Su Kim; Hye-Jeong Lee; Sang-Hyun Park; Yeu-Chun Kim; Jungoh Ahn

doi:10.1016/j.btre.2021.e00626

Expression and purification of soluble and active human enterokinase light chain in Escherichia coli

Biotechnol Rep (Amst). 2021 May 5:30:e00626. doi: 10.1016/j.btre.2021.e00626. eCollection 2021 Jun.

Authors

Young Su Kim^{1

2}, Hye-Jeong Lee², Sang-Hyun Park^{2

3}, Yeu-Chun Kim¹, Jungoh Ahn^{2

3}

Affiliations

¹ Department of Chemical and Biomolecular Engineering, KAIST, Daejeon 34141, Republic of Korea.
² Biotechnology Process Engineering Center, KRIBB, Cheongju 28116, Republic of Korea.
³ Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea.

Abstract

Human enterokinase light chain (hEK_L) specifically cleaves the sequence (Asp)₄-Lys↓X (D₄K), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEK_L production from Escherichia coli is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEK_L from E. coli by expressing the hEK_L variant C112S fused with maltose-binding protein (MBP) through D₄K and molecular chaperons including GroEL/ES. The fusion protein self-cleaved in vivo, thereby removing the MBP in the E. coli cells. Thus, the self-cleaved hEK_L variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEK_L variant exhibiting an enzymatic activity of 3.1 × 10³ U/mL (9.934 × 10⁵ U/mg). The approaches presented here greatly simplify the purification of hEK_L from E. coli without requiring refolding processes.

Keywords: D4K, (Aspartic Acid)4 Lysine; EK, enterokinase; Escherichia coli; Fusion technology; Human enterokinase light chain; IPTG, isopropyl β-D-1-thiogalactopyranoside; MBP, maltose-binding protein; Recombinant protein; Self-cleavage; TEV, tobacco etch virus; bEKL, bovine enterokinase light chain; hEKL, human enterokinase light chain.