RESUMO
cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.
