Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts
Antibodies have been used for basic research, clinical diagnostics, and therapeutic applications. <i>Escherichia coli</i> is one of the organisms of choice for the production of recombinant antibodies. Variable antibody genes have canonical and non-canonical disulfide bonds that are form...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-08-01
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| Series: | Antibodies |
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| Online Access: | https://www.mdpi.com/2073-4468/9/3/39 |
| _version_ | 1827711579111555072 |
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| author | Bhupal Ban Maya Sharma Jagathpala Shetty |
| author_facet | Bhupal Ban Maya Sharma Jagathpala Shetty |
| author_sort | Bhupal Ban |
| collection | DOAJ |
| description | Antibodies have been used for basic research, clinical diagnostics, and therapeutic applications. <i>Escherichia coli</i> is one of the organisms of choice for the production of recombinant antibodies. Variable antibody genes have canonical and non-canonical disulfide bonds that are formed by the oxidation of a pair of cysteines. However, the high-level expression of an antibody is an inherent problem to the process of disulfide bond formation, ultimately leading to mispairing of cysteines which can cause misfolding and aggregation as inclusion bodies (IBs). This study demonstrated that fragment antibodies are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies when expressed using engineered bacterial host strains with optimal culture conditions. It was observed that moderate-solubilization and an in vitro matrix that associated refolding strategies with redox pairing more correctly folded, structured, and yielded functionally active antibody fragments than the one achieved by a direct dilution method in the absence of a redox pair. However, natural antibodies have canonical and non-canonical disulfide bonds that need a more elaborate refolding process in the presence of optimal concentrations of chaotropic denaturants and redox agents to obtain correctly folded disulfide bonds and high yield antibodies that retain biological activity. |
| first_indexed | 2024-03-10T17:57:33Z |
| format | Article |
| id | doaj.art-07e472021ccb498bbb32aeff5cb76695 |
| institution | Directory Open Access Journal |
| issn | 2073-4468 |
| language | English |
| last_indexed | 2024-03-10T17:57:33Z |
| publishDate | 2020-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibodies |
| spelling | doaj.art-07e472021ccb498bbb32aeff5cb766952023-11-20T09:07:54ZengMDPI AGAntibodies2073-44682020-08-01933910.3390/antib9030039Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial HostsBhupal Ban0Maya Sharma1Jagathpala Shetty2Antibody Engineering and Technology Core, University of Virginia, Charlottesville, VA 22904, USADepartment of Data Science, School of Informatics and Computing Indiana University–Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USAAntibody Engineering and Technology Core, University of Virginia, Charlottesville, VA 22904, USAAntibodies have been used for basic research, clinical diagnostics, and therapeutic applications. <i>Escherichia coli</i> is one of the organisms of choice for the production of recombinant antibodies. Variable antibody genes have canonical and non-canonical disulfide bonds that are formed by the oxidation of a pair of cysteines. However, the high-level expression of an antibody is an inherent problem to the process of disulfide bond formation, ultimately leading to mispairing of cysteines which can cause misfolding and aggregation as inclusion bodies (IBs). This study demonstrated that fragment antibodies are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies when expressed using engineered bacterial host strains with optimal culture conditions. It was observed that moderate-solubilization and an in vitro matrix that associated refolding strategies with redox pairing more correctly folded, structured, and yielded functionally active antibody fragments than the one achieved by a direct dilution method in the absence of a redox pair. However, natural antibodies have canonical and non-canonical disulfide bonds that need a more elaborate refolding process in the presence of optimal concentrations of chaotropic denaturants and redox agents to obtain correctly folded disulfide bonds and high yield antibodies that retain biological activity.https://www.mdpi.com/2073-4468/9/3/39recombinant antibodiesinclusion bodiesrefoldingredox pairdisulfide bonds |
| spellingShingle | Bhupal Ban Maya Sharma Jagathpala Shetty Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts Antibodies recombinant antibodies inclusion bodies refolding redox pair disulfide bonds |
| title | Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts |
| title_full | Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts |
| title_fullStr | Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts |
| title_full_unstemmed | Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts |
| title_short | Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts |
| title_sort | optimization of methods for the production and refolding of biologically active disulfide bond rich antibody fragments in microbial hosts |
| topic | recombinant antibodies inclusion bodies refolding redox pair disulfide bonds |
| url | https://www.mdpi.com/2073-4468/9/3/39 |
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