title: Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.
abstract: We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Marcel
authors: M
authors: Bokhove
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Hamed
authors: H
authors: Sadat Al Hosseini
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Takako
authors: T
authors: Saito
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Elisa
authors: E
authors: Dioguardi
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Katharina
authors: K
authors: Gegenschatz-Schmid
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Kaoru
authors: K
authors: Nishimura
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Isha
authors: I
authors: Raj
authors: ESRF - The European Synchrotron, Grenoble 38000, France.
authors: Daniele
authors: D
authors: de Sanctis
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
authors: Ling
authors: L
authors: Han
authors: Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden. Electronic address: luca.jovine@ki.se.
authors: Luca
authors: L
authors: Jovine
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins
keywords: Crystallization; Glycoproteins; Maltose-binding protein fusion; Mammalian cell expression; Molecular replacement; X-ray crystallography