Scheme to identify enhanced peptide ligands of PSA epitope, HL10
A) Mutations were introduced into the HL10 epitope using degenerate oligonucleotides, which when expressed result in random amino acid substitutions. N represents any one of 4 bases; X represents any given amino acid codon. A library was generated for each position of the peptide epitope. B) Illustration of the oligonucleotides corresponding to the epitope region used to generate altered peptide ligand (APL) sequences. C) Sequences encoding APL products were cloned into the pQE40 vector and transformed en mass into E. coli. Ninety individual colonies were picked and arrayed into wells of a 96 well plate. This procedure was conducted for each position within the sequence of the HL10 epitope to generate the ten individual positional mutant libraries. The bacterial clones were subsequently expressed, lysed in individual wells of 96 well plate, and individually coupled to tosyl-activated magnetic beads that were then used as a source of antigen in the functional assay.