An improved method for the isolation and identification of unknown proteins that bind to known DNA sequences by affinity capture and mass spectrometry

Abstract

Transcription of a gene can be regulated at many different levels. One such fundamental level is interaction between protein and DNA. Protein(s) binds to distinct sites on the DNA, which activate, enhance or repress transcription. Despite being such an important process, very few tools exist to identify the proteins that interact with chromosome, most of which are in vitro in nature. Here, we propose an in vivo based method for identification of DNA binding protein(s) in bacteria where the DNA-protein complex formed in vivo is crosslinked by formaldehyde. This complex is further isolated and the bound proteins are identified. The method was used to isolate promoter DNA binding proteins, which bind and regulate the dsz operon in Gordonia sp. IITR 100 responsible for biodesulfurization of organosulfurs. The promoter binding proteins were identified by MALDI ToF MS/MS and the binding was confirmed by gel shift assay. Unlike other reported in vivo methods, this improved method does not require sequence of the whole genome or a chip and can be scaled up to improve yields.