Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

• Published in: Journal of structural biology Vol. 194; no. 1; pp. 1 - 7
• Main Authors: Bokhove, Marcel, Sadat Al Hosseini, Hamed, Saito, Takako, Dioguardi, Elisa, Gegenschatz-Schmid, Katharina, Nishimura, Kaoru, Raj, Isha, de Sanctis, Daniele, Han, Ling, Jovine, Luca
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2016; Academic Press
• Subjects: Amino Acid Sequence; Animals; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; CHO Cells; Cricetinae; Cricetulus; Crystallization; Crystallography, X-Ray; Gene Expression; Glycoproteins; HEK293 Cells; Humans; Maltose-binding protein fusion; Maltose-Binding Proteins - chemistry; Maltose-Binding Proteins - genetics; Maltose-Binding Proteins - metabolism; Mammalian cell expression; Medicin och hälsovetenskap; Models, Molecular; Molecular replacement; Mutation; Protein Conformation; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sf9 Cells; Technical Note; X-ray crystallography; VERL; ORF; Mammalian cell expression; IMAC; TEV; Endo H; Crystallization; PNGase F; UMOD; Glycoproteins; CHO; Molecular replacement; Crypα; HEK; PDB; Maltose-binding protein fusion; SEC; X-ray crystallography; PEI; TEVCS; MBP; mMBP; AKH; ENG
• ISSN: 1047-8477; 1095-8657; 1095-8657
• DOI: 10.1016/j.jsb.2016.01.016

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.