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

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Title
Expression and purification of soluble and active human enterokinase light chain in Escherichia coli
Author(s)
Young Su Kim; Hye Jeong Lee; Sang-Hyun Park; Y C Kim; Jungoh Ahn
Bibliographic Citation
Biotechnology Reports, vol. 30, pp. e00626-e00626
Publication Year
2021
Abstract
Human enterokinase light chain (hEKL) specifically cleaves the sequence (Asp)4-Lys↓X (D4K), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEKL production from Escherichia coli is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEKL from E. coli by expressing the hEKL 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 hEKL variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEKL 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 hEKL from E. coli without requiring refolding processes.
Keyword
Human enterokinase light chainEscherichia coliRecombinant proteinFusion technologySelf-cleavage
ISSN
2215-017X
DOI
http://dx.doi.org/10.1016/j.btre.2021.e00626
Type
Article
Appears in Collections:
Division of Bio Technology Innovation > BioProcess Engineering Center > 1. Journal Articles
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