title: Purification of coagulation factor VIII by immobilized metal affinity chromatography.
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 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.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Estela S
authors.initials: ES
authors.lastname: Rodrigues
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Claudia I
authors.initials: CI
authors.lastname: Verinaud
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Oliveira
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Isaías
authors.initials: I
authors.lastname: Raw
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Alexandre P Y
authors.initials: AP
authors.lastname: Lopes
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Elizabeth A L
authors.initials: EA
authors.lastname: Martins
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Cheng
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D005169:Factor VIII / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular
chemical_list: D003300:Copper; D005169:Factor VIII
keywords: anion exchange chromatography; coagulation factor VIII; immobilized metal affinity chromatography; plasma protein purification; vitamin-K-dependent coagulation factors