High-yield expression and purification of recombinant human macrophage migration inhibitory factor.
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.
Mozetic-Francky
B
B
BIOHIT d.o.o., Ljubljana, Slovenia.
Cotic
V
V
Ritonja
A
A
Zerovnik
E
E
Francky
A
A
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
D008263:Macrophage Migration-Inhibitory Factors; D011994:Recombinant Proteins