High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14.
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.
Hunter
M J
MJ
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Chazin
W J
WJ
D000595:Amino Acid Sequence; D000943:Antigens, Differentiation / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D040501:Calgranulin A; D040502:Calgranulin B; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019281:Dimerization; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D000943:Antigens, Differentiation; D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D040502:Calgranulin B; D018076:DNA, Complementary; D011994:Recombinant Proteins