Purification of coagulation factor VIII by immobilized metal affinity chromatography

Author(s)

Rodrigues, Estela S. ^[1] ; Verinaud, Claudia I. ^[1] ; Oliveira, Douglas S. ^[1] ; Raw, Isaias ^[1] ; Lopes, Alexandre P. Y. ^[1] ; Martins, Elizabeth A. L. ^[1] ; Cheng, Elisabeth ^[1]

Affiliation

^[1] Inst Butantan, Ctr Biotecnol, BR-05503900 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Biotechnology and Applied Biochemistry; v. 62, n. 3, p. 343-348, MAY-JUN 2015.
• Web of Science Citations: 6

Abstract

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 Cu2+ 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-Cu2+ column, and FVIII was eluted with 200mM 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 60mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column. (AU)

• FAPESP's process: 07/07764-5 - Purification of coagulation factor VIII and protein C from human plasma employing chromatographic methods
• Grantee: Estela de Souza Rodriguez
• Support Opportunities: Scholarships in Brazil - Scientific Initiation