Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic Purification
Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in <i>Escherichia coli</i> often leads to the formation of inclusion bodies. To address this issue, we devised and tested four differ...
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MDPI AG
2023-10-01
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| author | Kyu Tae Byun Boram Kim Junmin Cho Inbeom Lee Myung Gu Lee Dongsun Park Tae-Bong Kang Hyung-Sik Won Chan Gil Kim |
| author_facet | Kyu Tae Byun Boram Kim Junmin Cho Inbeom Lee Myung Gu Lee Dongsun Park Tae-Bong Kang Hyung-Sik Won Chan Gil Kim |
| author_sort | Kyu Tae Byun |
| collection | DOAJ |
| description | Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in <i>Escherichia coli</i> often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar <i>K</i><sub>d</sub>) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications. |
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| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T21:24:32Z |
| publishDate | 2023-10-01 |
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| series | Biomolecules |
| spelling | doaj.art-77e7b02ece7e475cbccdbd4bb51a76592023-11-19T15:50:14ZengMDPI AGBiomolecules2218-273X2023-10-011310150810.3390/biom13101508Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic PurificationKyu Tae Byun0Boram Kim1Junmin Cho2Inbeom Lee3Myung Gu Lee4Dongsun Park5Tae-Bong Kang6Hyung-Sik Won7Chan Gil Kim8Department of Biotechnology, Research Institute (RIBHS), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of KoreaDepartment of Biotechnology, Research Institute (RIBHS), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of KoreaDepartment of Biotechnology, Research Institute (RIBHS), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of KoreaDepartment of Biotechnology, Research Institute (RIBHS), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of KoreaKonkukbio Inc., Konkuk University, Chungju 27478, Republic of KoreaDepartment of Biology Education, Korea National University of Education, Cheongju 28173, Republic of KoreaDepartment of Biotechnology, Research Institute (RIBHS), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of KoreaDepartment of Biotechnology, Research Institute (RIBHS), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of KoreaDepartment of Biotechnology, Research Institute (RIBHS), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of KoreaAlthough single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in <i>Escherichia coli</i> often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar <i>K</i><sub>d</sub>) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.https://www.mdpi.com/2218-273X/13/10/1508antibodysingle-chain variable fragment (scFv)human epidermal growth factor receptor 2 (HER2)bacterial production<i>Escherichia coli</i>maltose-binding protein (MBP) |
| spellingShingle | Kyu Tae Byun Boram Kim Junmin Cho Inbeom Lee Myung Gu Lee Dongsun Park Tae-Bong Kang Hyung-Sik Won Chan Gil Kim Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic Purification Biomolecules antibody single-chain variable fragment (scFv) human epidermal growth factor receptor 2 (HER2) bacterial production <i>Escherichia coli</i> maltose-binding protein (MBP) |
| title | Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic Purification |
| title_full | Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic Purification |
| title_fullStr | Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic Purification |
| title_full_unstemmed | Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic Purification |
| title_short | Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in <i>Escherichia coli</i> and One-Step Chromatographic Purification |
| title_sort | development of an anti her2 single chain variable antibody fragment construct for high yield soluble expression in i escherichia coli i and one step chromatographic purification |
| topic | antibody single-chain variable fragment (scFv) human epidermal growth factor receptor 2 (HER2) bacterial production <i>Escherichia coli</i> maltose-binding protein (MBP) |
| url | https://www.mdpi.com/2218-273X/13/10/1508 |
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