Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.
Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.
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DOI：
10.1093/protein/gzaa015
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发表时间：
2019-12
期刊：
影响因子：
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通讯作者：
S. O’Rourke;G. I. Morozov;Jacob T. Roberts;A. Barb;N. Sgourakis
中科院分区：
文献类型：
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作者：
S. O’Rourke;G. I. Morozov;Jacob T. Roberts;A. Barb;N. Sgourakis
目前用于产生具有所需结合肽（pMHC-I）的主要组织相容性复合体（MHC）I类蛋白以用于研究、诊断和治疗应用的方法受到空MHC-I分子的固有不稳定性的限制。利用分子伴侣TAP结合蛋白相关蛋白（TAPBPR）的特性，我们开发了一种稳健的方法，在中国人卵巢细胞中以高产率生产可溶性肽受体MHC-I分子，完全绕过了大肠杆菌中表达的包涵体的费力重折叠的要求。纯化的MHC-I/TAPBPR复合物可以制备用于多种人类同种异型，并且在保守的Asn 86残基处表现出复杂的聚糖修饰。作为一个概念的证明，我们证明了两个相关的肿瘤相关抗原的T细胞HLA等位基因特异性肽结合和MHC限制性抗原识别。我们的系统提供了一种简便、高通量的方法，用于产生pMHC-I抗原，以探测和扩增多克隆T细胞库中存在的TCR特异性。
Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.