RIPPA: Identification of MHC‐II Binding Peptides from Antigen Using a Yeast Display‐Based Approach

Abstract

Mapping MHC-II binding peptides derived from an antigenic protein for potential CD4+ T-cell epitopes has been challenging due to a lack of experimental approaches that are both quantitative and rapid. The rate-limiting steps in current approaches include the construction of single MHC allele expressing cell lines and/or the purification of the MHC-II allelic proteins for peptide elution (i.e., mass spectrometry) or in vitro peptide binding (i.e., ELISA) assays. These labor-intensive steps typically take up to 4 months or more. In this protocol, we describe a system that uses yeast cells to display "empty" (i.e., without covalently linked peptides) MHC-II heterodimers that are capable of binding exogenously added peptides of interest. This yeast-MHC-II system eliminates the time-consuming soluble MHC-II purification steps, allowing rapid identification of peptide ligands from protein antigens (RIPPA). The amount of peptide loading to MHC-II or the extent of competition between indicator and competitor peptides at the surface of yeast cells can be quantitatively determined using flow cytometric analysis. Importantly, the protocol only takes ∼1 month from the construction of plasmids and the yeast display of "empty" MHC-II to the quantitative determination of MHC-II binding peptides from a given antigen.

TCR-like antibodies targeting autoantigen-mhc complexes: a mini-review

“…Combining phage display with yeast display is particularly useful for developing high affinity TCRL mAbs ( 71 ). Since first developed ( 42 ), yeast display technology has evolved, allowing surface display of monomeric or dimeric protein scaffolds ( 72 , 73 ). Thus, either scFv or Fab identified from a phage-Ab library can be affinity matured using the yeast platform.…”