HIGH-YIELD EXPRESSION AND PURIFICATION OF RECOMBINANT HUMAN MACROPHAGE-MIGRATION INHIBITORY FACTOR

We have expressed the human macrophage migration inhibitory factor (MI F) in Escherichia coli using the pKP 1500 expression plasmid, which co ntains the tac promoter and a temperature-sensitive origin of replicat ion, to ensure a high plasmid copy number at elevated temperatures. Th e 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. O ne gram of recombinant human MIF was isolated from 50 g of E. coli cel ls (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 ac tivity. The near-UV CD and the H-1 NMR spectra confirmed its highly or dered, native-like structure. The far-UV CD spectrum revealed that rec ombinant human MIF contains well-defined secondary structure. (C) 1997 Academic Press.