Journal
BIOTECHNOLOGY PROGRESS
Volume 27, Issue 5, Pages 1206-1217Publisher
WILEY
DOI: 10.1002/btpr.637
Keywords
human alpha-galactosidase A; Fabry's disease; recombinant protein; transient gene expression; mammalian cells
Funding
- MICINN [EUI2008-03610, ERANET-IB 08-007]
- AGAUR [2009SGR-108]
- Fundacio Marato TV3
- CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN)
- CIBER de Enfermedades Raras (CIBERER)
- VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program
- Instituto de Salud Carlos III
- European Regional Development Fund
- ICREA ACADEMIA (Catalonia, Spain)
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Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human alpha-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 x His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1206-1217, 2011
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