Stability engineering, biophysical, and biological characterization of the myeloid activating receptor immunoglobulin-like transcript 1 (ILT1/LIR-7/LILRA2)
Stability engineering, biophysical, and biological characterization of the myeloid activating receptor immunoglobulin-like transcript 1 (ILT1/LIR-7/LILRA2)
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骨髓激活受体免疫球蛋白样转录物 1 (ILT1/LIR-7/LILRA2) 的稳定性工程、生物物理和生物学表征
DOI：
10.1016/j.pep.2007.08.010
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发表时间：
2007-12-01
影响因子：
1.6
通讯作者：
Gao, George F.
中科院分区：
文献类型：
--
作者：
Chen, Yong;Chu, Fuliang;Gao, George F.
免疫球蛋白样转录物1（ILT1/LIR-7/LILRA2/CD85h）是ILT家族中的活化受体之一，其成员已被报道调节参与免疫应答的广泛细胞。虽然抑制性ILT受体已被广泛研究，然而，ILT激活受体的功能和结构尚未阐明。获得足够量的重组蛋白是对给定蛋白进行功能和结构研究的必要条件。ILT1的胞外区在重组生产过程中容易形成聚集体，这是研究的一个技术瓶颈。在这里，我们报告了通过定点诱变（R142C）工程化大规模稳定生产ILT1 DID2结构域，所述定点诱变在氨基酸位置142处引入半胱氨酸以与备用cys132形成二硫键，而不受基于同源ILT 2/4/11的已知结构的天然蛋白质的拓扑影响。重组ILT1 D1D2结构域在溶液中表现为稳定的二聚体和单体的平衡，并产生用于结构测定的理想晶体。大量可溶性ILT1 DID2结构域的获得为进一步研究其详细结构和功能提供了有用的试剂。(C)2007年爱思唯尔公司All rights reserved.
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 DID2 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 DID2 domains provides useful reagent for further studies of its detailed structure and functions. (C) 2007 Elsevier Inc. All rights reserved.