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Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells
Author(s) -
Barton William A.,
TzvetkovaRobev Dorothea,
ErdjumentBromage Hediye,
Tempst Paul,
Nikolov Dimitar B.
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.062244206
Subject(s) - computational biology , recombinant dna , yeast , structural biology , biology , chemistry , microbiology and biotechnology , biochemistry , gene
The advent of the multiwavelength anomalous diffraction phasing method has significantly accelerated crystal structure determination and has become the norm in protein crystallography. This method allows researchers to take advantage of the anomalous signal from diverse atoms, but the dominant method for derivative preparation is selenomethionine substitution. Several generally applicable, high‐efficiency labeling protocols have been developed for use in the bacterial, yeast, and baculovirus/insect cell expression systems but not for mammalian tissue culture. As a large number of proteins of biomedical importance can only be produced in yields sufficient for X‐ray diffraction experiments in mammalian expression systems, it becomes all the more important to develop such protocols. We therefore evaluated several variables that play roles in determining incorporation levels and report here a simple protocol for selenomethionine modification of proteins in mammalian cells routinely yielding >90% labeling efficiency.