Extended Data Fig. 3: Development of HLA-B*27:05 yeast library.
From: Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides
a. GRb TCR tetramer staining of wildtype SRY-β2M-HLA-B*27:05 single chain trimer expressed on yeast surface. Single chain trimer expression was monitored by anti-C-Myc-488. Streptavidin-647 (SA-647) was used to tetramerize and label the GRb TCR. b. Schematic of the SRY-β2m-HLA-B*27:05 single chain trimer on yeast surface. Error prone PCR introduced random mutations in SRY peptide, β2m and HLA-B*27:05 heavy chain. Sequencing of the enriched SRY-β2M-HLA-B*27:05 error prone library identified mutations located on HLA-B*27:05 heavy chain, shown as cartoon diagram and colored green. Bona fide mutations are shown as stick diagram and colored red. c. GRb TCR tetramer staining was rescued by M5L, H114Y and A153D mutation of the SRY-β2m-HLA-B*27:05 single chain trimer (HLA-B*27:053mut). Single chain trimer expression was monitored by anti-C-Myc-488. Streptavidin-647 (SA-647) was used to tetramerize and label the GRb TCR. d. Location of M5L, H114Y, and A153D mutations shown on the HLA-B*27 ribbon structure. e. Library design shows the anchor residue preference for the HLA-B*27:053mut library at the P2 and PΩ. For other positions, the NNK codon allowed all 20 amino acids and stop codons. Nucleotide abbreviations for codon usage are listed according to the IUPAC nucleotide code. Library diversities were estimated based on SDCAA plate colony numbers under serial dilution. f. GRb TCR tetramer staining of 4th round peptide-HLA-B*27:053mut yeast-display library, indicating successful enrichment. g. Deep sequencing counts on the most enriched peptides after 4 rounds of selection with the GRb TCR. h. WebLogo plots of the top 100 peptides after 4th round of selection with the GRb TCR, the two predominant clusters within the selection, and the sequence of the cognate peptide recognized by the GRb TCR in the abundance at the given position among the unique peptides.
