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 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.
Mancia
Filippo
F
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
Patel
Saurabh D
SD
Rajala
Michael W
MW
Scherer
Philipp E
PE
Nemes
Adriana
A
Schieren
Ira
I
Hendrickson
Wayne A
WA
Shapiro
Lawrence
L
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D006729:Hormones, Ectopic / Q000096:biosynthesis*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis*; D051379:Mice; D051381:Rats; D011985:Receptors, Serotonin / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D052243:Resistin
D006729:Hormones, Ectopic; D008565:Membrane Proteins; C499624:RETN protein, human; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D052243:Resistin; C499623:Retn protein, mouse; C499622:Retn protein, rat; D049452:Green Fluorescent Proteins