title: Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells.
abstract: 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.
authors.affiliation: Department of Biochemistry, Virginia Commonwealth University, Richmond, 23298, USA. wabarton@vcu.edu
authors.forename: William A
authors.initials: WA
authors.lastname: Barton
authors.forename: Dorothea
authors.initials: D
authors.lastname: Tzvetkova-Robev
authors.forename: Hediye
authors.initials: H
authors.lastname: Erdjument-Bromage
authors.forename: Paul
authors.initials: P
authors.lastname: Tempst
authors.forename: Dimitar B
authors.initials: DB
authors.lastname: Nikolov
mesh_terms: D019943:Amino Acid Substitution; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D003470:Culture Media; D006801:Humans; D042787:Receptor, TIE-2 / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D012645:Selenomethionine; D042787:Receptor, TIE-2