title: Glycoprotein structural genomics: solving the glycosylation problem.
abstract: Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.
authors.affiliation: Nuffield Department of Clinical Medicine and MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.
authors.forename: Veronica T
authors.initials: VT
authors.lastname: Chang
authors.forename: Max
authors.initials: M
authors.lastname: Crispin
authors.forename: A Radu
authors.initials: AR
authors.lastname: Aricescu
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Nettleship
authors.forename: Janet A
authors.initials: JA
authors.lastname: Fennelly
authors.forename: Chao
authors.initials: C
authors.lastname: Yu
authors.forename: Kent S
authors.initials: KS
authors.lastname: Boles
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Evans
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
mesh_terms: D000470:Alkaloids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors; D023281:Genomics / Q000379:methods; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D017026:Swainsonine / Q000737:chemistry / Q000378:metabolism
chemical_list: D000470:Alkaloids; D004791:Enzyme Inhibitors; D006023:Glycoproteins; C065629:kifunensine; D017026:Swainsonine