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首页> 外文期刊>Journal of Molecular Biology >Directed Evolution of Soluble Single-chain Human Class II MHC Molecules.
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Directed Evolution of Soluble Single-chain Human Class II MHC Molecules.

机译：可溶性单链人类II类MHC分子的定向进化。

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Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present antigenic peptides to T cells. Expression of these molecules in soluble form has met limited success, presumably due to their large size, heterodimeric structure and the presence of multiple disulfide bonds. Here we have used directed evolution and yeast surface display to engineer soluble single-chain human lymphocyte antigen (HLA) class II MHC DR1 molecules without covalently attached peptides (scDR1alphabeta). Specifically, a library of mutant scDR1alphabeta molecules was generated by random mutagenesis and screened by fluorescence activated cell sorting (FACS) with DR-specific conformation-sensitive antibodies, yielding three well-expressed and properly folded scDR1alphabeta variants displayed on the yeast cell surface. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo indicated three amino acid residues in the beta1 domain are important for the improved protein folding yield. Further, molecular modeling studies suggested these mutations might increase the protein folding efficiency by improving the packing of a hydrophobic core in the alpha1beta1 domain of DR1. The scDR1alphabeta mutants displayed on the yeast cell surface are remarkably stable and bind specifically to DR-specific peptide HA(306-318) with high sensitivity and rapid kinetics in flow cytometric assays. Moreover, since the expression, stability and peptide-binding properties of these mutants can be directly assayed on the yeast cell surface using immuno-fluorescence labeling and flow cytometry, time-consuming purification and refolding steps of recombinant DR1 molecules are eliminated. Therefore, these scDR1alphabeta molecules will provide a powerful technology platform for further design of DR1 molecules with improved peptide-binding specificity and affinity for therapeutic and diagnostic applications. The methods described here should be generally applicable to other class II MHC molecules and also class I MHC molecules for their functional expression, characterization and engineering.
机译：主要的组织相容性复合物（MHC）II类分子是将抗原性肽呈递给T细胞的膜锚定异二聚体。这些分子以可溶形式的表达获得了有限的成功，大概是由于它们的大尺寸，异二聚体结构和多个二硫键的存在。在这里，我们已经使用定向进化和酵母表面展示技术来工程化可溶性单链人淋巴细胞抗原（HLA）II类MHC DR1分子，而没有共价连接的肽（scDR1alphabeta）。具体而言，通过随机诱变生成突变体scDR1alphabeta分子的文库，并通过DR特异性构象敏感抗体通过荧光激活细胞分选（FACS）进行筛选，从而在酵母细胞表面显示出三个表达良好且折叠正确的scDR1alphabeta变体。对这些进化的变体和从头产生的一些定点突变体的详细分析表明，beta1域中的三个氨基酸残基对提高蛋白质折叠率非常重要。此外，分子建模研究表明，这些突变可能通过改善DR1的alpha1beta1域中疏水核心的堆积而提高蛋白质折叠效率。酵母细胞表面上显示的scDR1alphabeta突变体非常稳定，并在流式细胞仪检测中以高灵敏度和快速动力学特异性结合DR特异性肽HA（306-318）。而且，由于可以使用免疫荧光标记和流式细胞术直接在酵母细胞表面上测定这些突变体的表达，稳定性和肽结合特性，因此省去了重组DR1分子的耗时的纯化和重折叠步骤。因此，这些scDR1alphabeta分子将为进一步设计具有改进的肽结合特异性和亲和力的DR1分子提供强大的技术平台，以用于治疗和诊断应用。此处描述的方法通常应适用于其他II类MHC分子，以及I类MHC分子的功能表达，特征描述和工程改造。

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