Expression and purification of soluble and active human enterokinase light chain in Escherichia coli
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 prese...
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| Format: | Article |
| Language: | English |
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Elsevier
2021-06-01
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| Series: | Biotechnology Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215017X21000424 |
| _version_ | 1818737333813379072 |
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| author | Young Su Kim Hye-Jeong Lee Sang-hyun Park Yeu-chun Kim Jungoh Ahn |
| author_facet | Young Su Kim Hye-Jeong Lee Sang-hyun Park Yeu-chun Kim Jungoh Ahn |
| author_sort | Young Su Kim |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-18T00:51:23Z |
| format | Article |
| id | doaj.art-d9db4e83a3724c138b02dd86cd5ee74a |
| institution | Directory Open Access Journal |
| issn | 2215-017X |
| language | English |
| last_indexed | 2024-12-18T00:51:23Z |
| publishDate | 2021-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biotechnology Reports |
| spelling | doaj.art-d9db4e83a3724c138b02dd86cd5ee74a2022-12-21T21:26:40ZengElsevierBiotechnology Reports2215-017X2021-06-0130e00626Expression and purification of soluble and active human enterokinase light chain in Escherichia coliYoung Su Kim0Hye-Jeong Lee1Sang-hyun Park2Yeu-chun Kim3Jungoh Ahn4Department of Chemical and Biomolecular Engineering, KAIST, Daejeon 34141, Republic of Korea; Biotechnology Process Engineering Center, KRIBB, Cheongju 28116, Republic of KoreaBiotechnology Process Engineering Center, KRIBB, Cheongju 28116, Republic of KoreaBiotechnology Process Engineering Center, KRIBB, Cheongju 28116, Republic of Korea; Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of KoreaDepartment of Chemical and Biomolecular Engineering, KAIST, Daejeon 34141, Republic of Korea; Corresponding author.Biotechnology Process Engineering Center, KRIBB, Cheongju 28116, Republic of Korea; 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; Corresponding author at: Biotechnology Process Engineering Center, KRIBB, Cheongju 28116, Republic of Korea.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.http://www.sciencedirect.com/science/article/pii/S2215017X21000424Human enterokinase light chainEscherichia coliRecombinant proteinFusion technologySelf-cleavage |
| spellingShingle | Young Su Kim Hye-Jeong Lee Sang-hyun Park Yeu-chun Kim Jungoh Ahn Expression and purification of soluble and active human enterokinase light chain in Escherichia coli Biotechnology Reports Human enterokinase light chain Escherichia coli Recombinant protein Fusion technology Self-cleavage |
| title | Expression and purification of soluble and active human enterokinase light chain in Escherichia coli |
| title_full | Expression and purification of soluble and active human enterokinase light chain in Escherichia coli |
| title_fullStr | Expression and purification of soluble and active human enterokinase light chain in Escherichia coli |
| title_full_unstemmed | Expression and purification of soluble and active human enterokinase light chain in Escherichia coli |
| title_short | Expression and purification of soluble and active human enterokinase light chain in Escherichia coli |
| title_sort | expression and purification of soluble and active human enterokinase light chain in escherichia coli |
| topic | Human enterokinase light chain Escherichia coli Recombinant protein Fusion technology Self-cleavage |
| url | http://www.sciencedirect.com/science/article/pii/S2215017X21000424 |
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