Discovery of protein interactions using parallel analysis of translated ORFs (PLATO)

Abstract

Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.

Figure 1

Parallel analysis of in vitro translated ORFs (PLATO). (a) ORF display scheme. The pooled human ORFeome v5.1 entry vector library is is attL-attR (“LR”) recombined into the pRD-DEST expression vector. Expression plasmids are PCR amplified to generate the DNA templates for in vitro transcription. Following in vitro translation, the protein-mRNA-ribosome complexes are incubated with protein, antibody or small-molecule bait immobilized on beads. The enriched mRNA library is recovered from bait-prey bead complexes for further analysis. (b) Processing of mRNA samples for deep DNA sequencing. After fragmentation and reverse transcription (RT) using a universal primer to recover the 3′ end of ORFeome transcripts, cDNA is polyadenylated with terminal deoxynucleotide transferase (TdT) and amplified for multiplex deep sequencing using primers containing a sample barcode and the P5 and P7 Illumina sequencing adaptors. (c) Sequencing reads of the unenriched human pRD-ORFeome mRNA library (the ‘input’ library). Most ORFs were sequenced at least once.

Figure 2

Identification of known and previously undescribed interactions using PLATO. (a) Interactions with LYN tyrosine-protein kinase. Scatter plot of each ORF’s sequencing reads after enrichment on GST-LYN or GST. Several known and undescribed LYN binding candidates are highlighted in red. (b) Enrichment of two known interactors of LYN. Data were normalized to the GST enriched libraries (n=3, mean ± s.d.; *, P < 0.01; t test). (c) Confirmation of known and predicted LYN binding proteins by affinity precipitation-western blotting of lysates from HEK 293T cells transiently overexpressing the individual V5-His-tagged candidate proteins. (d) Confirmation of previously unidentified autoantigens from a PND patient. (e) Interactions with autoantibodies. Enrichment ranking of PND autoantigens identified using CSF from patient C. (f) Interactions with a small molecule. Enrichment of previously identified targets of gefitinib. Data were normalized to the control-enriched libraries (n=3, mean ± s.d.; *, P < 0.05; t test).