TF-interactome by HaloTag protein arrays

What is it about?

We applied an improved NAPPA methodology, HaloTag-NAPPA, to map hormone response TF interactions. The TF interactome network that was so mapped illuminates previously unknown cross-regulation of signaling pathways regulated by multiple TFs. The HaloTag-NAPPA assay provides a complementary approach to high throughput interactome mapping with HT-Y2H or co-complex mass-spec methods

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Why is it important?

HaloTag-NAPPA arrays present several advantages over conventional protein arrays. The synthesis and capture of proteins in situ eliminates the cumbersome process of expressing and purifying these proteins and hence avoids compromising protein functional integrity. Additionally, because DNA is spotted instead of proteins, the arrays require no special storage conditions and have an extended shelf-life of greater than 12 months. HaloTag fusion proteins irreversibly bind to a small chloroalkane ligand that is co-spotted on the array. This approach yields rapid and high affinity capture of expressed fusion proteins with minimal lateral protein diffusion. We also developed an alternative protein expression technique to express proteins on the high density HaloTag-NAPPA arrays. The submerge protocol most likely prevented local diffusion effects by alleviating the space constraints on protein synthesis that were previously imposed by the small Hybrigasket incubation chambers, allowing non-captured Halo-fused proteins to be moved away from the site of synthesis with gentle agitation