TY - JOUR
T1 - Optimization of protein production in mammalian cells with a coexpressed fluorescent marker
AU - Mancia, Filippo
AU - Patel, Saurabh D.
AU - Rajala, Michael W.
AU - Scherer, Philipp E.
AU - Nemes, Adriana
AU - Schieren, Ira
AU - Hendrickson, Wayne A.
AU - Shapiro, Lawrence
N1 - Funding Information:
We are grateful to Richard Axel for helpful discussions and support throughout the course of this work, Hubi Amrein and Thomas Livelli for carrying out the majority of the work involved in the construction of the pFM vectors, and for their generosity in allowing us their use. We thank Peter Chen for help with bioinformatics analysis of PDB structures and Paul Lee for helpful discussions. F.M. was supported by fellowships from the European Molecular Biology Organization and the Human Frontiers Science Program. This work was supported by grants from NIH (GM68671, W.A.H. and F.M.; GM68671 and GM62529, L.S.; R01-DK55758, P.E.S.; Medical Scientist Training Grant T32-GM97288, M.W.R.), and grants from the American Diabetes Association and Foundation for Research to Prevent Blindness (L.S.). Part of this work was performed within the New York Structural Genomics Research Consortium (NYSGXRC).
PY - 2004/8
Y1 - 2004/8
N2 - 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.
AB - 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.
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U2 - 10.1016/j.str.2004.06.012
DO - 10.1016/j.str.2004.06.012
M3 - Comment/debate
C2 - 15296729
AN - SCOPUS:4143053500
SN - 0969-2126
VL - 12
SP - 1355
EP - 1360
JO - Structure
JF - Structure
IS - 8
ER -