Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins

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art_DEMASI_Enhanced_Proteolytic_Processing_of_Recombinant_Human_Coagulation_Factor_2016.PDF (1.01 MB)
Citações na Scopus
3
Tipo de produção
article
Data de publicação
2016
DOI
Scopus
Web of Science
PubMed
Título da Revista
ISSN da Revista
Título do Volume
Editora
HUMANA PRESS INC
Autores
MOLINA, Erika de S.
BOWMAN-COLIN, Christian
LOJUDICE, Fernando H.
MURAS, Angelita
SOGAYAR, Mari C.
Citação
MOLECULAR BIOTECHNOLOGY, v.58, n.6, p.404-414, 2016
Projetos de Pesquisa
Unidades Organizacionais
Fascículo
Resumo
Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.
Palavras-chave
Coagulation factor VIII, Proteolytic processing, PACE, PCSK5, PCSK6, PCSK7
URI
https://observatorio.fm.usp.br/handle/OPI/14561
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Coleções
Artigos e Materiais de Revistas Científicas - HC/Outros