title: Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization.
abstract: Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.
authors.affiliation: Molecular Microbiology and Microbial Pathogenesis Program, Washington University School of Medicine; Department of Internal Medicine, Washington University School of Medicine; Drug Discovery Program in Pulmonary and Critical Care Medicine, Washington University School of Medicine.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Kober
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine; Drug Discovery Program in Pulmonary and Critical Care Medicine, Washington University School of Medicine; Biochemistry Program, Washington University School of Medicine; Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Yurtsever
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine; Drug Discovery Program in Pulmonary and Critical Care Medicine, Washington University School of Medicine; Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine; Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine; Department of Cell Biology and Physiology, Washington University School of Medicine; TBRETT@dom.wustl.edu.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Brett
mesh_terms: D000470:Alkaloids / Q000737:chemistry; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection / Q000191:economics / Q000379:methods*
chemical_list: D000470:Alkaloids; D006023:Glycoproteins; D009942:Organometallic Compounds; D011994:Recombinant Proteins; C065629:kifunensine; C088321:nickel nitrilotriacetic acid; D011094:Polyethyleneimine; D009571:Nitrilotriacetic Acid