Purification of coagulation factor VIII by immobilized metal affinity chromatography
| Autor: | Douglas S. Oliveira, Estela S. Rodrigues, Claudia Iwashita Verinaud, Isaias Raw, Elizabeth A. L. Martins, Alexandre P. Y. Lopes, Elisabeth Cheng |
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| Rok vydání: | 2014 |
| Předmět: |
chemistry.chemical_classification
congenital hereditary and neonatal diseases and abnormalities Chromatography Elution Process Chemistry and Technology Biomedical Engineering Bioengineering General Medicine Ligand (biochemistry) Applied Microbiology and Biotechnology Metal Sepharose chemistry.chemical_compound Adsorption chemistry Coagulation hemic and lymphatic diseases visual_art Drug Discovery visual_art.visual_art_medium Molecular Medicine Imidazole Glycoprotein Biotechnology |
| Zdroj: | Biotechnology and Applied Biochemistry. 62:343-348 |
| ISSN: | 0885-4513 |
| DOI: | 10.1002/bab.1276 |
| Popis: | Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column. |
| Databáze: | OpenAIRE |
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