title: Eukaryotic expression: developments for structural proteomics.
abstract: The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
authors.affiliation: Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, Oxford, England.
authors.forename: A R
authors.initials: AR
authors.lastname: Aricescu
authors.forename: R
authors.initials: R
authors.lastname: Assenberg
authors.forename: R M
authors.initials: RM
authors.lastname: Bill
authors.forename: D
authors.initials: D
authors.lastname: Busso
authors.forename: V T
authors.initials: VT
authors.lastname: Chang
authors.forename: S J
authors.initials: SJ
authors.lastname: Davis
authors.forename: A
authors.initials: A
authors.lastname: Dubrovsky
authors.forename: L
authors.initials: L
authors.lastname: Gustafsson
authors.forename: K
authors.initials: K
authors.lastname: Hedfalk
authors.forename: U
authors.initials: U
authors.lastname: Heinemann
authors.forename: I M
authors.initials: IM
authors.lastname: Jones
authors.forename: D
authors.initials: D
authors.lastname: Ksiazek
authors.forename: C
authors.initials: C
authors.lastname: Lang
authors.forename: K
authors.initials: K
authors.lastname: Maskos
authors.forename: A
authors.initials: A
authors.lastname: Messerschmidt
authors.forename: S
authors.initials: S
authors.lastname: Macieira
authors.forename: Y
authors.initials: Y
authors.lastname: Peleg
authors.forename: A
authors.initials: A
authors.lastname: Perrakis
authors.forename: A
authors.initials: A
authors.lastname: Poterszman
authors.forename: G
authors.initials: G
authors.lastname: Schneider
authors.forename: T K
authors.initials: TK
authors.lastname: Sixma
authors.forename: J L
authors.initials: JL
authors.lastname: Sussman
authors.forename: G
authors.initials: G
authors.lastname: Sutton
authors.forename: N
authors.initials: N
authors.lastname: Tarboureich
authors.forename: T
authors.initials: T
authors.lastname: Zeev-Ben-Mordehai
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D040901:Proteomics / Q000379:methods*; D012645:Selenomethionine; D015003:Yeasts / Q000378:metabolism
chemical_list: D012645:Selenomethionine