Rapid interactome profiling by massive sequencing

Abstract

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.

Figure 1.

Interactome discovery pipeline. Poly A⁺ RNA is fragmented and retro-transcribed to cDNA by random priming. After normalization and linker ligation cDNAs are cloned into pPAO10 ORF-filtering vector and the library is filtered on chloramphenicol/ampicillin containing plates. After CRE-mediated recombination, ORF-displaying phages are challenged with TG2 throughout two cycles of selection and amplification. The ORF inserts of selected phages are obtained by digestion with restriction fragments and massively sequenced. Reads are mapped to the human genome and identified genomic regions are ranked on the basis of read coverage. Clones containing candidate interacting ORFs are rescued from selected libraries by inverse PCR using primers matching the contig sequence core. Recovered clones are expressed and validated by ELISA.

Figure 2.

(A) Rank abundance curve obtained by plotting the total number of mapped informative reads (presence of both 454 primers) versus the total number of identified genes. (B) Chart shows how many genes are supported by different number of reads. As a result of cDNA normalization the vast majority of genes are represented by up to 10 reads while only very few genes show more reads (with a maximum of 680). X-axis has been limited to 50.

Figure 3.

(A) Venn diagram of the first 50 genes on the ranking list of non selected (NS) and selected (BIO, SP) libraries. (B) Probability of identifying all top five or top 10 genes by random selecting sample of clones of increasing size. Probability (pTop) has been approximated by a 10 000 cycle simulation for each sample size (picked clones). Simulation was performed using a custom developed PERL script. (C) Focus indexes for the first 50 genes of each library ranked on the basis of the number of supporting reads (coverage). The horizontal bars represent the mean value.

Figure 4.

ELISA reactivity of the top five clones from BIO and SP selection. Reactivity was tested on recombinant human and mouse TG2 proteins and on negative control proteins (BSA and WNT4) coated on solid surface (A) and as soluble biotinylated proteins (B).