title: Expression and purification of cytokine receptor homology domain of human granulocyte-colony-stimulating factor receptor fusion protein in Escherichia coli.
abstract: Direct expression of the cytokine receptor homology (CRH) domain of granulocyte-colony-stimulating factor (G-CSF) receptor is lethal to Escherichia coli. For the efficient and stable production of an active CRH domain in E. coli, we fused the CRH domain with different proteins, such as maltose-binding protein (MalE), glutathione S-transferase, and thioredoxin (Trx). Among these, Trx appeared to be the best in terms of the protein expression level, purification efficiency by affinity chromatography, and binding activity to its ligand, G-CSF. The yield of active Trx-CRH fusion protein increased about 200-fold compared to that of previously reported MalE-CRH fusion.
authors.affiliation: Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Tatsuda
authors.forename: H
authors.initials: H
authors.lastname: Arimura
authors.forename: H
authors.initials: H
authors.lastname: Tokunaga
authors.forename: M
authors.initials: M
authors.lastname: Ishibashi
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
authors.forename: M
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D018528:ATP-Binding Cassette Transporters; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor; D006801:Humans; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D018121:Receptors, Cytokine / Q000737:chemistry*; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013879:Thioredoxins / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D018121:Receptors, Cytokine; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D016179:Granulocyte Colony-Stimulating Factor; D013879:Thioredoxins; D005982:Glutathione Transferase; D003545:Cysteine