Expression,production, and renaturation of a functional single-chain
variable antibody fragment (scFv) against human ICAM-1 |
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| Authors: | H. Sun G.M. Wu Y.Y. Chen Y. Tian Y.H. Yue G.L. Zhang |
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| Affiliation: | 1. Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China;2. Department of Biological Pharmacy, Heilongjiang Vocational College of Biology Science and Technology, Harbin, China;3. Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China;4. College of Animal Husbandry and Veterinary Medicine, Jilin University, Changchun, China |
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| Abstract: | Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progressionof inflammatory responses in vivo. To develop a newanti-inflammatory drug to block the biological activity of ICAM-1, we produced amonoclonal antibody (Ka=4.19×10−8 M) against humanICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressedat a high level as inclusion bodies in Escherichia coli. We refoldedthe scFv (Ka=2.35×10−7 M) by ion-exchange chromatography,dialysis, and dilution. The results showed that column chromatography refolding byhigh-performance Q Sepharose had remarkable advantages over conventional dilution anddialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higherwith this method. The purity of the final product was greater than 90%, as shown bydenaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, andanimal experiments were used to assess the immunological properties and biologicalactivities of the renatured scFv. |
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| Keywords: | Intercellular adhesion molecule-1 Single-chain variable antibody fragment Expression Purification Renaturation Biological activity |
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