Stability engineering, biophysical, and biological characterization of the myeloid activating receptor immunoglobulin-like transcript 1 (ILT1/LIR-7/LILRA2).
Immunoglobulin-like transcript 1 (ILT1/LIR-7/LILRA2/CD85h) is one of the activating receptors in the ILT family whose members have been reported to regulate a broad range of cells involved in the immune response. Although inhibitory ILT receptors have been extensively studied, however, functions and structures of ILT activating receptors have yet to be elucidated. Obtaining of sufficient amount of recombinant proteins is a requisite for the functional and structural studies of a given protein. As a technical bottleneck of the study, extracellular domains of the ILT1 form aggregation during recombinant production in the past efforts. Here, we report the large-scale stable production of ILT1 D1D2 domains through engineering of site-directed mutagenesis (R142C) that introduces a cysteine at amino acid position 142 to form a disulfide bond with the spare cys132 without topological influences of the native protein based on the known structures of the homologous ILT 2/4/11. The recombinant ILT1 D1D2 domains behave as an equilibrium of both stable dimer and monomer in solution and yield ideal crystals for structural determination. The availability of quantities of soluble ILT1 D1D2 domains provides useful reagent for further studies of its detailed structure and functions.
Chen
Yong
Y
China-Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Chu
Fuliang
F
Gao
Feng
F
Zhou
Bin
B
Gao
George F
GF
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D019281:Dimerization; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C105298:LILRA2 protein, human; D008024:Ligands; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D001710:Biotin