title: High-throughput production of human proteins for crystallization: the SGC experience.
abstract: Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of approximately 40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9-20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.
authors.affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Oxford, UK. pavel.savitsky@sgc.ox.ac.uk
authors.forename: Pavel
authors.initials: P
authors.lastname: Savitsky
authors.forename: James
authors.initials: J
authors.lastname: Bray
authors.forename: Christopher D O
authors.initials: CD
authors.lastname: Cooper
authors.forename: Brian D
authors.initials: BD
authors.lastname: Marsden
authors.forename: Pravin
authors.initials: P
authors.lastname: Mahajan
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Burgess-Brown
authors.forename: Opher
authors.initials: O
authors.lastname: Gileadi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011506:Proteins; D011994:Recombinant Proteins