Optimization of Protein Production in Mammalian Cells with a Coexpressed Fluorescent Marker

The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure...

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Bibliographic Details
Published in:Structure (London) 2004-08, Vol.12 (8), p.1355-1360
Main Authors: Mancia, Filippo, Patel, Saurabh D, Rajala, Michael W, Scherer, Philipp E, Nemes, Adriana, Schieren, Ira, Hendrickson, Wayne A, Shapiro, Lawrence
Format: Article
Language:English
Subjects:
Animals
Blotting, Western
Cells, Cultured
Flow Cytometry
Green Fluorescent Proteins - biosynthesis
Hormones, Ectopic - biosynthesis
Humans
Membrane Proteins - biosynthesis
Mice
Rats
Receptors, Serotonin - biosynthesis
Recombinant Proteins - biosynthesis
Recombinant Proteins - metabolism
Resistin
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Summary:The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure for the direct selection of stable mammalian cell lines that express proteins of interest in high yield. Coexpression of a marker protein, such as green fluorescent protein, is linked to that of the desired protein through an internal ribosome entry site in the vector that is transfected into cells in culture. The coexpressed marker is used to select for highly expressing clonal cell lines. Applications are described to a membrane protein, the 5HT2c serotonin receptor, and to a secreted cysteine-rich protein, resistin. Besides providing an expeditious means for producing mammalian proteins for structural work, the resulting cell lines also readily support tests of functional properties and structure-inspired hypotheses.
ISSN:0969-2126
1878-4186
DOI:10.1016/j.str.2004.06.012