Total number of peptides identified from common proteins in the T cell and B cell self-presentation repertoire.

<p>Experiments were performed in triplicate (for T cell clones) and in duplicate (for B cell lines) using cells prepared from donor 1 (mean±range).</p

Self-peptides identified from the cell-type specific proteome include key signaling receptors such as CD3, CD4 and the B cell receptor.

<p><b>A</b>. Cell-type specific nested peptide sequences isolated from T cells and B cells (donor 1). Predicted core binding sequence is indicated in yellow. <b>B</b>. Frequency of peptides derived from the common (on non-cell-type specific) proteome and the cell-type specific proteome in T cells and B cells. Experiments were performed in triplicate (for T cell clones) and in duplicate (for B cell lines) using cells prepared from donor 1 (mean±SD).</p

Self-peptides identified in two donors with unique class II MHC expression.

<p>Peptides with identical core sequences isolated from both donor 1 and donor 2 are indicated in bold.</p

Number and frequency of peptides identified.

a<p>% of total sequences.</p>b<p>% of cell-type specific sequences.</p

Core sequence of a subset of abundant self-peptides isolated from both T cells and B cells (donor 1).

<p>The core sequence represents the largest consensus peptide sequence identified for total nested protein fragments across all samples for a single donor. The total number of peptides identified for donor 1 that contain each core sequence is indicated. Predicted p1 anchor residues for DRB1 binding are in bold (p1 = Y, F, L, I, V, W).</p

Isolation of HLA-DR:self-peptide complexes from an expanded class II MHC positive T cell clone (T cell) and a EBV-transformed B cell clone from the same donor.

<p><b>A</b>. Characterization of class II MHC, class I MHC, and CD4 expression in a representative genetically-matched T cell clone and B cell line. <b>B</b>. Strategy used to identify self-peptides. HLA-DR:peptide complexes were immunoprecipitated from T and B cell lysates. Peptides were eluted from the MHC and assessed for quality and contaminants with MALDI before being subjected to multidimensional protein sequencing and identification using the SEQUEST algorithm. <b>C</b>. Frequency of common self-peptides identified T and B cells from a donor 1 (DRB1*1301; DRB1*1501 DRB3*0202; DRB5*0101).</p

Predicted class II MHC occupancy for self-peptides derived from a single donor with multiple MHC alleles.

<p>Peptides with both weak (<500 nM) and strong (<50 nM) predicted affinity are considered as positive for binding. The large frequency of peptides for which no MHC binding could be predicted or which greater than 3 MHC alleles was predicted suggests the likelihood of MHC promiscuity among self-peptides.</p

Orthogonal Labeling of M13 Minor Capsid Proteins with DNA to Self-Assemble End-to-End Multiphage Structures

M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices

Identification of new aerolysin receptors.

<p>^BiotinAeL.CP was used to identify new GPI-anchored proteins that bind Aerolysin<b>. A</b> Biotin.LPETG was attached to the N-terminus of proaerolysin via sortagging. The purified reaction product was analyzed by immunoblot. <b>B</b> HeLa cells were incubated with ^BiotinAeL.CP for 3 hours at 4°C and subsequently lysed with 0.5% NP-40. After pull-down with neutravidin beads, proteins were eluted, analyzed by SDS-PAGE, and subjected to mass spectrometry. Five GPI-anchored proteins were identified. UniProt accession codes are indicated. Peptides identified by mass spectrometry, lipidated amino acids, signal peptides, as well as peptides cleaved off from the pro-proteins are highlighted. <b>C</b> Binding of ^BiotinAeL.CP to mesothelin and to CD59 was verified by immunoblot.</p

Strategies for site-specific labeling of proaerolysin.

<p><b>A</b> Structure of the proaerolysin monomer (PDB: 1PRE). Proaerolysin consists of several different domains, two of which are responsible for receptor binding (domains 1 and 2), one containsing the trans-membrane domain, and the C-terminal peptide (CP), which functions as a chaperone and dissociates from the rest of the complex upon heptamer association and pore formation. <b>B</b> Sortase reaction mechanism. C-terminal sortagging: sortase cleaves after threonine in the context of its recognition motif resulting in the formation of a new covalent bond with the N-terminus of an added oligoglycine or oligoalanine nucleophile coupled to a label of choice. N-terminal sortagging: the N-terminal glycine of proaerolysin is recognized as a nucleophile by sortase and conjugated to an LPXTG/A probe bearing a label. <b>C</b> Structures of probes used in this study. Not depicted is AAA.Alexa Fluor 647, which is similar to GGG.Alexa Fluor 647, but with alanine replacing glycine. PelB: periplasm targeting sequence, cleaved off by the producer bacteria upon export of proaerolysin to the periplasm. H6: hexahistidine handle for affinity purification. Protease cleavage sites are recognized by target cell surface proteases such as furin. CP: C-terminal peptide, serves as a chaperone for proaerolysin. Upon its loss, proaerolysin is converted to mature aerolysin (AeL). <b>D</b> Scheme for wild type (WT) and sortaggable versions of proaerolysin with their designations. The LPXTG/A pentapeptides are sortase recognition motifs.</p