New Approach for Studying of Isoforms and High-Homology Proteins in Mammalian Cells.

ABSTRACT

In mammals, a large number of proteins are expressed as more than one isoform, resulting in the increased diversity of their proteome. Understanding the functions of isoforms is very important, since individual isoforms of the same protein can have oncogenic or pathogenic properties, or serve as disease markers. The high homology of isoforms with ubiquitous expression makes it difficult to study them. In this work, we propose a new approach for the study of protein isoforms in mammalian cells, which makes it possible to individually detect and investigate the functions of an individual isoform. The approach was developed to study the functions of isoforms of the PHF10 protein, a chromatin subunit of the PBAF remodeling complex. We demonstrated the possibility of induced simultaneous suppression of all endogenous PHF10 isoforms and the expression of a single recombinant FLAG-tagged isoform. For this purpose, we created constructs based on the pSLIK plasmid with a cloned cassette containing the recombinant gene of interest and miR30 with the corresponding shRNAs. The doxycycline-induced activation of the cassette allows on and off switching. Using this construct, we achieved the preferential expression of only one recombinant PHF10 isoform with a simultaneously reduced number of all endogenous isoforms. Our approach can be used to study the role of point mutations, the functions of individual domains and important sites, or to individually detect untagged isoforms with knockdown of all endogenous isoforms.