ABSTRACT
Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid-induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence. All four probes revealed an acid-denatured state below pH 3 which resembled a typical molten globule. The pH transition is not two-state as the CD data at 222 nm deviated from all other probes. Urea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an apparent DeltaGU app H2O of 31 +/- 3 kJ.mol-1 when extrapolated to zero denaturant concentration. However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentration (in 10-fold range) even though size-exclusion chromatogryphy (SEC) revealed a dimer-monomer dissociation prior to MIF unfolding. Refolding experiments were performed starting from acid, guanidinium-Cl and urea-denatured states. The kinetics were multiphasic with at least two folding intermediates. The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s-1 (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol-1.
Subject(s)
Macrophage Migration-Inhibitory Factors/chemistry , Protein Folding , Circular Dichroism , Escherichia coli , Guanidine/chemistry , Humans , Hydrogen-Ion Concentration , Kinetics , Protein Denaturation , Recombinant Proteins/chemistry , Urea/chemistryABSTRACT
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.
