Production of recombinant human factor VIII in different cell lines and the effect of human XBP1 co-expression.
Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.
Campos-da-Paz
Mariana
M
Departamento de Biologia Celular-IB, Laboratório de Biologia Molecular, Universidade de Brasília, 70910-900, Brasilia, DF, Brazil.
Costa
Christiane Silva
CS
Quilici
Luana Salgado
LS
de Carmo Simões
Isabella
I
Kyaw
Cynthia Maria
CM
Maranhão
Andrea Queiroz
AQ
Brigido
Marcelo Macedo
MM
D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D005169:Factor VIII / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000096:biosynthesis; D006801:Humans; D009687:Nuclear Proteins / Q000096:biosynthesis*; D010957:Plasmids / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D014162:Transfection / Q000379:methods*; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C078147:F8 protein, human; D005169:Factor VIII