High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14.

Hunter MJ, Chazin WJ
J Biol Chem. 1998 273 (20): 12427-35

PMID: 9575199 · DOI:10.1074/jbc.273.20.12427

The phenotypical and functional heterogeneity of different macrophage subpopulations are defined by discrete changes in the expression of two S100 calcium-binding proteins, migration inhibitory factor-related proteins (MRPs) 8 and 14. To further our understanding of MRP8 and MRP14 in the developmental stages of inflammatory responses, overexpression of the MRPs was obtained through a combination of a T7-based expression vector and the Escherichia coli BL21 (DE3) cell line. An efficient, two-step chromatographic protocol was then developed for rapid, facile purification. Extensive biophysical characterization and chemical cross-linking experiments show that MRP8 and MRP14 form oligomers with a strong preference to associate as a heterodimer. Heteronuclear NMR experiments indicate that a specific well packed dimer is formed only in equimolar mixtures of the two proteins. Our results suggest that there is a unique complementarity in the interface of the MRP8/MRP14 complex that cannot be fully reproduced in the MRP8 and MRP14 homodimers.

MeSH Terms (15)

Amino Acid Sequence Antigens, Differentiation Calcium-Binding Proteins Calgranulin A Calgranulin B Circular Dichroism Cloning, Molecular Dimerization DNA, Complementary Magnetic Resonance Spectroscopy Molecular Sequence Data Mutagenesis Recombinant Proteins Sequence Homology, Amino Acid Spectrometry, Fluorescence

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