Bacterial Production of Recombinant Coagulation Factor VIII Domains
Factor VIII (F8) is a blood coagulation protein prearranged in six domains, and its deficiency causes hemophilia A. To fashion functional F8 therapeutics, development of a recombinant F8 (rF8) domain is essential not only for F8 substitution, but also to decipher the F8-related mechanisms. In this s...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-04-01
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| Series: | Medicina |
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| Online Access: | https://www.mdpi.com/1648-9144/59/4/694 |
| _version_ | 1797604361857138688 |
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| author | Saima Bashar Hee-Jin Jeong |
| author_facet | Saima Bashar Hee-Jin Jeong |
| author_sort | Saima Bashar |
| collection | DOAJ |
| description | Factor VIII (F8) is a blood coagulation protein prearranged in six domains, and its deficiency causes hemophilia A. To fashion functional F8 therapeutics, development of a recombinant F8 (rF8) domain is essential not only for F8 substitution, but also to decipher the F8-related mechanisms. In this study, we generated Glutathione S-transferase (GST)-conjugated recombinant A2 and A3 domains of F8 using <i>Escherichia coli</i>. The high growth rate and economically advantageous protein production system in terms of inexpensive reagents and materials in <i>E. coli</i> cells facilitated the completion of entire process from protein expression to purification in 3–4 days with low production cost. Subsequent assessment of these purified proteins using enzyme-linked immunosorbent assay (ELISA) and antibodies against F8 revealed enhanced detection of rF8-A2 or rF8-A3 in a concentration dependent manner, indicating the presence of the antibody-binding epitopes in these proteins. Furthermore, these proteins are suitable for generating novel antibodies against the F8 domain and F8 domain-capturing affinity columns by enabling their conjugation to GST-capturing beads. Additionally, the recombinant F8 domains produced herein can be used for various studies, which include investigating the explicit roles of the F8 domain in the coagulation process, with domain-specific binding partners, and antibodies. |
| first_indexed | 2024-03-11T04:46:16Z |
| format | Article |
| id | doaj.art-253fc3ef0fbc47c2aadbf74dc75662ad |
| institution | Directory Open Access Journal |
| issn | 1010-660X 1648-9144 |
| language | English |
| last_indexed | 2024-03-11T04:46:16Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Medicina |
| spelling | doaj.art-253fc3ef0fbc47c2aadbf74dc75662ad2023-11-17T20:20:38ZengMDPI AGMedicina1010-660X1648-91442023-04-0159469410.3390/medicina59040694Bacterial Production of Recombinant Coagulation Factor VIII DomainsSaima Bashar0Hee-Jin Jeong1Industry-Academia Cooperation Foundation, Hongik University, 2639 Sejong-ro, Sejong-si 30016, Republic of KoreaDepartment of Biological and Chemical Engineering, Hongik University, 2639 Sejong-ro, Sejong-si 30016, Republic of KoreaFactor VIII (F8) is a blood coagulation protein prearranged in six domains, and its deficiency causes hemophilia A. To fashion functional F8 therapeutics, development of a recombinant F8 (rF8) domain is essential not only for F8 substitution, but also to decipher the F8-related mechanisms. In this study, we generated Glutathione S-transferase (GST)-conjugated recombinant A2 and A3 domains of F8 using <i>Escherichia coli</i>. The high growth rate and economically advantageous protein production system in terms of inexpensive reagents and materials in <i>E. coli</i> cells facilitated the completion of entire process from protein expression to purification in 3–4 days with low production cost. Subsequent assessment of these purified proteins using enzyme-linked immunosorbent assay (ELISA) and antibodies against F8 revealed enhanced detection of rF8-A2 or rF8-A3 in a concentration dependent manner, indicating the presence of the antibody-binding epitopes in these proteins. Furthermore, these proteins are suitable for generating novel antibodies against the F8 domain and F8 domain-capturing affinity columns by enabling their conjugation to GST-capturing beads. Additionally, the recombinant F8 domains produced herein can be used for various studies, which include investigating the explicit roles of the F8 domain in the coagulation process, with domain-specific binding partners, and antibodies.https://www.mdpi.com/1648-9144/59/4/694coagulation factor disordersfactor VIIIrecombinant proteinhemophilia A |
| spellingShingle | Saima Bashar Hee-Jin Jeong Bacterial Production of Recombinant Coagulation Factor VIII Domains Medicina coagulation factor disorders factor VIII recombinant protein hemophilia A |
| title | Bacterial Production of Recombinant Coagulation Factor VIII Domains |
| title_full | Bacterial Production of Recombinant Coagulation Factor VIII Domains |
| title_fullStr | Bacterial Production of Recombinant Coagulation Factor VIII Domains |
| title_full_unstemmed | Bacterial Production of Recombinant Coagulation Factor VIII Domains |
| title_short | Bacterial Production of Recombinant Coagulation Factor VIII Domains |
| title_sort | bacterial production of recombinant coagulation factor viii domains |
| topic | coagulation factor disorders factor VIII recombinant protein hemophilia A |
| url | https://www.mdpi.com/1648-9144/59/4/694 |
| work_keys_str_mv | AT saimabashar bacterialproductionofrecombinantcoagulationfactorviiidomains AT heejinjeong bacterialproductionofrecombinantcoagulationfactorviiidomains |