Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) production

作者：Federico Mufarrege Eduardo; Antuna Sebastian; Etcheverrigaray Marina; Kratje Ricardo; Prieto Claudio

来源：Protein Expression and Purification, 2014, 95: 50-56.

DOI：10.1016/j.pep.2013.11.005

• Lentiviral vector
• rhFVIII production
• Protein overexpression
• CMV enhancer/promoter
• EF-1 alpha promoter

Background: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 5'UTR (5' untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells.
Results: By combining the human cytomegalovirus (CMV) or the elongation factor 1 alpha (EF-1 alpha) promoters along with different 5'UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 5'UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1 alpha promoter in three widely used cell lines.
Conclusion: In the present work, LVs containing different promoters and 5'UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.

• 出版日期2014-3