Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens (RIPPA)

Liu, Rongzeng; Jiang, Wei; Mellins, Elizabeth

doi:10.1038/s41423-021-00717-5

Cited by 15 publications

(29 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It contains the bi-directional GAL1-10 promoter to direct simultaneous expression of the α and β chains of the DR1 ectodomain. Because of the potential instability of the "empty" DQ6 ectodomain, we included leucine zipper (LZ) motifs to facilitate dimerization (Liu et al, 2021). The DQ6α chain with the Fos motif was located upstream of the yeast AGA2 gene and followed by an HA epitope tag, while the β chain with the Jun motif was designed to be secreted from yeast.…”

Section: Construction Of Yeast Shuttle Vector Expressing "Empty" Mhc-iimentioning

confidence: 99%

“…The peptide competition assay described in Basic Protocol 2 is adaptable to high throughput format (Liu et al, 2021). This alternative protocol provides an example using a 96well PCR plate to rapidly identify peptide ligands derived from a protein antigen (RIPPA).…”

Section: Rippa In a 96-well Formatmentioning

confidence: 99%

“…The protocol described above takes advantage of a single‐cell eucaryotic surface display system—yeast display—to express MHC‐II on the cell surface of yeast and determine the relative binding of tested peptides to MHC‐II, without the necessity of MHC‐II protein purification (Jiang & Boder, 2010; Liu et al., 2021). Here, we include step‐by‐step protocols for this purification‐free method for rapid identification of MHC‐II peptide ligands derived from protein antigens (RIPPA), which we have applied to identify immunogenic epitopes derived from the narcolepsy‐related hypocretin protein and the SARS‐CoV‐2 spike protein (Liu et al., 2021). The entire protocol takes ∼1 month, which significantly expedites a process (cell line preparation, MHC‐II purification, ELISA) that requires ∼4 months.…”

Section: Commentarymentioning

confidence: 99%

“…To allow peptide exchange, the linker (containing an enzyme recognition site) in between CLIP and MHC‐II is cleaved prior to the addition of exogenous peptides. The turnaround time, from the design of recombinant MHC‐II construct, the construction of insect or mammalian cell line, and the purification of soluble MHC‐II, to the cleavage of the covalent linker and the peptide loading, typically takes up to 4 months (Liu et al., 2021).…”

Section: Introductionmentioning

confidence: 99%

“…The peptide binding approach described in this protocol uses yeast cells to display “empty” MHC‐II heterodimers that are capable of binding exogenously added peptides (data in Liu et al., 2021). This yeast display system eliminates the step of MHC‐II purification and is appropriate for the rapid identification of peptide ligands from protein antigens (RIPPA).…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Liu

Jiang

et al. 2022

Current Protocols

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Construction Of Yeast Shuttle Vector Expressing "Empty" Mhc-iimentioning

confidence: 99%

Section: Rippa In a 96-well Formatmentioning

confidence: 99%