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.

Deep sequencing analysis of phage libraries using Illumina platform

“…The binding of phage library to a target, or "panning", narrows the naïve library of 10 9 clones to 10 5 -10 6 clones. This is a typical number of phage clones recovered after one round of panning, but only some of these clones have affinity for the target.…”

“…Arap, Pasqualini and co-workers were the first to use 454 sequencing to analyze ~50,000 sequences from a library of 7-mer peptides; the author applied this technology to identify peptides emerging from panning against different organs in vivo [5]. Subsequently, the same sequencing was used by several groups to monitor selection of binding proteins from a library of open reading frames (ORF) displayed on phage [6,7]. Sidhu and co-workers used 454-sequencing to boost selection of peptides binding to different PDZ domains [8][9][10].…”

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

“…This work stems from previous studies in which we succeeded in profiling the interactomes of single or complex mixtures of proteins 23,24 using a platform that combines ORF phage display library selection with NGS analysis. Encouraged by these results, we decided to exploit the power of this platform for revealing RNA-protein interactions.…”

“…[21][22][23] We believe this is because only fragments of functional ORFs can form foldable domains that do not adversely affect the folding and activity of the fused b-lactamase reporter protein. Random ORFs do not fold into coherent domains and lead to the aggregation, misfolding and inactivation of the folding reporter.…”

“…When combined with nextgeneration sequencing (NGS), as a way to analyze their complexity, these libraries are used as universal reagents that can be screened for several activities, including rapid interactome profiling. 23,24 In accordance with this assumption, we wanted to evaluate whether such an approach could add power to the field of RNA-protein interaction discovery.…”