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Purification of coagulation factor VIII by immobilized metal affinity chromatography
Author(s) -
Rodrigues Estela S.,
Verinaud Claudia I.,
Oliveira Douglas S.,
Raw Isaías,
Lopes Alexandre P. Y.,
Martins Elizabeth A. L.,
Cheng Elisabeth
Publication year - 2014
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1276
Subject(s) - chemistry , chromatography , affinity chromatography , elution , coagulation , sepharose , ligand (biochemistry) , biochemistry , enzyme , psychology , receptor , psychiatry
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.