Human Cells as Platform to Produce Gamma-Carboxylated Proteins
The gamma-carboxylated proteins belong to a family of proteins that depend on vitamin K for normal biosynthesis. The major representative gamma-carboxylated proteins are the coagulation system proteins, for example, factor VII, factor IX, factor X, prothrombin, and proteins C, S, and Z. These molecules have harbored posttranslational modifications, such as glycosylation and gamma-carboxylation, and for this reason they need to be produced in mammalian cell lines. Human cells lines have emerged as the most promising alternative to the production of gamma-carboxylated proteins. In this chapter, the methods to generate human cells as a platform to produce gamma-carboxylated proteins, for example the coagulation factors VII and IX, are presented. From the cell line modification up to the vitamin K adaptation of the produced cells is described in the protocols presented in this chapter.
Key wordsGamma-carboxylated proteins Vitamin K cycle Human cell lines Coagulation factors Recombinant proteins
The authors acknowledge São Paulo Research Foundation—FAPESP (2015/19017-6), Conselho Nacional de Pesquisa—CNPq (142406/2016-3), Centro de Pesquisa, Inovação e Difusão (CEPID), and National Institute of Science and Technology in Stem Cell and Cell Therapy—INCTC for financial support and Sandra Navarro for drawing the figures.
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