title: High-yield expression and purification of recombinant human macrophage migration inhibitory factor.
abstract: We have expressed the human macrophage migration inhibitory factor (MIF) in Escherichia coli using the pKP 1500 expression plasmid, which contains the tac promoter and a temperature-sensitive origin of replication, to ensure a high plasmid copy number at elevated temperatures. The recombinant protein accumulated intracellularly in soluble form. We have designed a simple two-step procedure for protein purification by gel filtration on Sephadex G-50 and cation exchange chromatography on CM cellulose columns. This results in significantly improved yields. One gram of recombinant human MIF was isolated from 50 g of E. coli cells (wet weight). The 12.5-kDa protein was shown to be pure by SDS-PAGE, IEF, and HPLC. The identity of the purified protein was verified by N-terminal amino acid sequencing. The purified protein exhibits MIF activity. The near-UV CD and the 1H NMR spectra confirmed its highly ordered, native-like structure. The far-UV CD spectrum revealed that recombinant human MIF contains well-defined secondary structure.
authors.affiliation: BIOHIT d.o.o., Ljubljana, Slovenia.
authors.forename: B
authors.initials: B
authors.lastname: Mozetic-Francky
authors.forename: V
authors.initials: V
authors.lastname: Cotic
authors.forename: A
authors.initials: A
authors.lastname: Ritonja
authors.forename: E
authors.initials: E
authors.lastname: Zerovnik
authors.forename: A
authors.initials: A
authors.lastname: Francky
mesh_terms: D000818:Animals; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006168:Guinea Pigs; D006801:Humans; D007525:Isoelectric Focusing; D008263:Macrophage Migration-Inhibitory Factors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D017421:Sequence Analysis
chemical_list: D008263:Macrophage Migration-Inhibitory Factors; D011994:Recombinant Proteins