Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Sciences Center, Abilene, TX 79601, United States.
Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Sciences Center, Abilene, TX 79601, United States.
J Immunol Methods. 2019 Jan;464:22-30. doi: 10.1016/j.jim.2018.10.006. Epub 2018 Oct 19.
MHC class I-specific reagents such as fluorescently-labeled multimers (e.g., tetramers) have greatly advanced the understanding of CD8+ T cells under normal and diseased states. However, recombinant MHC class I components (comprising MHC class I heavy chain and β2 microglobulin) are usually produced in bacteria following a lengthy purification protocol that requires additional non-covalent folding steps with exogenous peptide for complete molecular assembly. We have provided an alternative and rapid approach to generating soluble and fully-folded MHC class I molecules in eukaryotic cell lines (such as CHO cells) using a Sleeping Beauty transposon system. Importantly, this method culminates in generating stable cell lines that reliably secrete epitope-defined MHC class I molecules into the tissue media for convenient purification and eventual biotinylation/multimerization. Additionally, MHC class I components are covalently linked, providing the opportunity to produce a diverse set of CD8+ T cell-specific reagents bearing peptides with various affinities to MHC class I.
MHC I 类特异性试剂，如荧光标记的多聚体（例如四聚体），极大地促进了对正常和患病状态下 CD8+T 细胞的理解。然而，重组 MHC I 类成分（包括 MHC I 类重链和β2 微球蛋白）通常在细菌中产生，需要经过冗长的纯化方案，其中需要与外源肽进行额外的非共价折叠步骤，以完成完整的分子组装。我们提供了一种替代方法，可在真核细胞系（如 CHO 细胞）中使用 Sleeping Beauty 转座子系统生成可溶性和完全折叠的 MHC I 类分子。重要的是，该方法最终生成了稳定的细胞系，可将表位定义的 MHC I 类分子可靠地分泌到组织培养基中，便于进行纯化和最终的生物素化/多聚化。此外，MHC I 类成分通过共价键连接，为生成具有各种 MHC I 类亲和力的肽的多样化 CD8+T 细胞特异性试剂提供了机会。
