IN VITRO FOLDING AND ASSEMBLY OF MHC MOLECULES

该项目的目标一直是开发体外系统，用于 研究MHC I类分子的组装和折叠。过去时 研究主要集中在体外转录/翻译耦合方面。 用于检查这三个链的组装的系统 大分子：I类重链、β-2微球蛋白和 抗原肽。最近关于体外突变体的结果强调了 这样的系统在评估装配参数方面的价值。我们目前的情况 方法是开发更多生物化学定义的方法，基于 经基因工程、细菌产生的单链MHC I类 蛋白质和合成抗原肽。我们已经证明了 具有生物活性、免疫刺激作用的MHC-I类多肽复合体可以 由完全还原和变性的体外复性制成 分子，这表明这是一种可行的方法来生产大型 实验用均相络合物的规模量。

The aim of this project has been to develop in vitro systems for the study of the assembly and folding of the MHC class I molecules. Past studies have focused on coupled in vitro transcription/translation systems for examination of the assembly of the three chains of this macromolecule: the class I heavy chain, beta-2 microglobulin, and antigenic peptide. Recent results with in vitro mutants emphasize the value of such a system in evaluating parameters of assembly. Our current approach is to develop more biochemically defined methods, based on genetically engineered, bacterially produced single chain MHC class I proteins and synthetic antigenic peptides. We have demonstrated that biologically active, immunostimulatory MHC class I peptide complexes can be made by in vitro refolding of completely reduced and denatured molecules, suggesting that this is a feasible approach to produce large scale amounts of homogenous complexes for experimental use.