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• W2016145428 abstract "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." @default.
• W2016145428 created "2016-06-24" @default.
• W2016145428 creator A5014465828 @default.
• W2016145428 creator A5054944661 @default.
• W2016145428 date "2004-06-01" @default.
• W2016145428 modified "2023-10-15" @default.
• W2016145428 title "Directed Evolution of Soluble Single-chain Human Class II MHC Molecules" @default.
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• W2016145428 doi "https://doi.org/10.1016/j.jmb.2004.04.054" @default.
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• W2016145428 hasPublicationYear "2004" @default.
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