Purification of coagulation factor VIII and protein C from human plasma employing chromatographic methods

• Grant number: 07/07764-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2008
• End date: July 31, 2008
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Elisabeth Cheng
• Grantee: Estela de Souza Rodriguez
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

In this project we intend to continue developing processes for the purification of coagulation factor VIII from human plasma. We propose two strategies that allow us to purify Protein C after the separation of factor VIII. We employ in these processes only chromatoghaphic methods,in contrast to the traditional methods used for plasma protein purifications, in which the processes start with the separation of crio-precopitate. Nowadays, it is possible to apply plasma directly to commercially available resins and, therefore, the use of huge centrifuges and refrigerated rooms can be avoided. In the first strategy, we propose to start the process with an ion exchange column to concentrate the proteins of interest. Based on previous results, we know that a greate deal of the plasma proteins can be eliminated in this step. Then, we employ an afinity resin specific for serine proteases. In this resin, protein C, which is a zimogen of serine proteases, will be attached to resin, while factor VIII will not. Finally, protein C will be separated from the other vitamin K dependent proteins in an IMAC column, since this protein possesses 15 exposed histidines. In the second proposed strategy, the process begins with a gel filtration to separate factor VIII from the low molecular weight proteins. Then, protein C containing fractions of the gel filtration are pooled and applied to an IMAC column.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

RODRIGUES, ESTELA S.; VERINAUD, CLAUDIA I.; OLIVEIRA, DOUGLAS S.; RAW, ISAIAS; LOPES, ALEXANDRE P. Y.; MARTINS, ELIZABETH A. L.; CHENG, ELISABETH. Purification of coagulation factor VIII by immobilized metal affinity chromatography. Biotechnology and Applied Biochemistry, v. 62, n. 3, p. 343-348, MAY-JUN 2015. (07/07764-5)