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

Autor:	Luca Jovine, Elisa Dioguardi, Isha Raj, Marcel Bokhove, Katharina Gegenschatz-Schmid, K. Nishimura, Hamed Sadat Al Hosseini, Takako Saito, Daniele de Sanctis, Ling Han
Rok vydání:	2016
Předmět:	PEI polyethylenimine Models Molecular 0301 basic medicine ENG endoglin/CD105 Maltose-binding protein fusion Protein Conformation Gene Expression Crystallography X-Ray Maltose-binding protein IMAC immobilized metal affinity chromatography Protein structure VERL vitelline envelope receptor for lysin Structural Biology Cricetinae Technical Note Sf9 Cells Peptide sequence chemistry.chemical_classification biology Chinese hamster ovary cell PNGase F peptide N-glycosidase F Biochemistry Crystallization UMOD uromodulin SEC size-exclusion chromatography Recombinant Fusion Proteins MBP maltose-binding protein CHO Cells CHO Chinese hamster ovary mMBP mammalianized maltose-binding protein Maltose-Binding Proteins TEV tobacco etch virus HEK human embryonic kidney 03 medical and health sciences Cricetulus ORF open reading frame Bacterial Proteins Affinity chromatography PDB Protein Data Bank Animals Humans Molecular replacement Amino Acid Sequence Glycoproteins X-ray crystallography TEVCS tobacco etch virus protease cleavage site Base Sequence 030102 biochemistry & molecular biology AKH adipokinetic hormone HEK 293 cells Endo H Endoglycosidase H Crystallography HEK293 Cells 030104 developmental biology chemistry Crypα cell adhesion molecule-like tyrosine phosphatase α Mammalian cell expression Mutation biology.protein Glycoprotein
Zdroj:	Journal of Structural Biology
ISSN:	1047-8477
DOI:	10.1016/j.jsb.2016.01.016
Popis:	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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5a4c8bcbb1b36465f3ae53db997e15f1 https://doi.org/10.1016/j.jsb.2016.01.016 Zobrazit plný text záznamu Full Text from ScienceDirect