Our genome encodes ~1800 predicted transcription factors (TFs). However, we do not have biochemical or functional data for the majority of these TFs. Hence, the development of tools to allow for the large-scale study of TFs, such as possible RNA- or protein-binding activity and post-translational modifications (PTMs), would be immensely valuable.
To date, proteins used in protein microarray technology has primarily been produced in Saccharomyces cerevisiae (Baker’s Yeast) with fewer instances of insect cell or in vitro coupled transcription-translation-based production. Expression of human proteins in these heterologous systems can result in the production of incorrectly-folded proteins. To address this, we have chosen to express these TFs in cultured human cells instead — with the view that these cells would give the proteins the best chance of folding correctly.
As a proof-of-concept, we have set up a workflow to clone, express, purify and array 50 TFs in a scalable medium-to-high throughput manner. With our array of 50 TFs, we have demonstrated that the arrayed TFs can bind to DNA — indicating that they are correctly folded. We have also successfully acetylated the TFs in vitro using purified CBP; demonstrating the utility of the arrays to study PTMs. We are now in the process of scaling up our array to 600 TFs.
We believe that the general workflow we have established can be applicable not only to TFs but to most proteins in the proteome.