Expression, purification, and refolding of the myeloid inhibitory receptor leukocyte immunoglobulin-like receptor-5 for structural and ligand identification studies.
The leukocyte immunoglobulin-like receptors (LIRs, also known as ILTs, CD85, and LILRs) comprise a family of related immunoregulatory receptors encoded within the leukocyte receptor cluster (LRC) on human chromosome 19. LIRs are transmembrane proteins containing either two or four extracellular immunoglobulin domains, and most family members are expressed predominantly on myeloid cell lineages. Although the inhibitory receptors LIR-1 and LIR-2 are known to bind to a broad range of class I MHC molecules and are thought to play important roles in immune regulation, the majority of LIRs are currently of unknown structure and their ligands remain unidentified. In this study, we describe recombinant production and characterisation of the extracellular portion of LIR-5 (ILT3), a poorly understood inhibitory receptor that transduces tolerising signals to dendritic cells. The two extracellular immunoglobulin domains of LIR-5 were expressed in Escherichia coli to a high level and were found to accumulate in inclusion bodies. Inclusion bodies were purified, solubilised, and receptor then renatured by dilution refolding, with acceptable yields. Size exclusion chromatography and SDS-PAGE analyses confirmed the extracellular portion behaved as a monomer in solution, and purified protein was antibody-reactive. LIR-5 is representative of a subset of LIR receptors that on the basis of structural and sequence comparisons with LIR-1 seem unlikely to bind class I MHC molecules. Successful prokaryotic generation of correctly folded LIR-5 in high levels has implications for production of other LRC receptors and should greatly facilitate attempts to define the structure and ligands of this important regulator of dendritic cell function.
Garner
Lee I
LI
Cancer Research UK Institute for Cancer Studies, University of Birmingham, Edgbaston, UK.
Salim
Mahboob
M
Mohammed
Fiyaz
F
Willcox
Benjamin E
BE
D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D003713:Dendritic Cells / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005805:Genes, MHC Class I / Q000502:physiology; D006801:Humans; D007108:Immune Tolerance / Q000502:physiology; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D008024:Ligands; D005810:Multigene Family / Q000502:physiology; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D015398:Signal Transduction / Q000502:physiology; D040681:Structural Homology, Protein; D020349:Surface Plasmon Resonance; D008562:Membrane Glycoproteins
D015703:Antigens, CD; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D008024:Ligands; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C105299:LILRB1 protein, human; C107907:LILRB4 protein, human; D008562:Membrane Glycoproteins