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Integrated approach to produce a recombinant, his‐tagged human α‐galactosidase a in mammalian cells
Author(s) -
Corchero José Luis,
Mendoza Rosa,
Lorenzo Julia,
RodríguezSureda Victor,
Domínguez Carmen,
Vázquez Esther,
FerrerMiralles Neus,
Villaverde Antonio
Publication year - 2011
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.637
Subject(s) - recombinant dna , transfection , enzyme , cell culture , biochemistry , in vitro , expression vector , rendering (computer graphics) , chemistry , specific activity , hek 293 cells , gene , biology , microbiology and biotechnology , computational biology , computer science , genetics , computer graphics (images)
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 α‐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 × 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. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011