Overproduction and purification of the Escherichia coli transcription factors “toxic” to a bacterial cell

• Published in: Protein expression and purification 2019-09, Vol.161, p.70-77
• Main Authors: Bessonova, Tatiana A., Lekontseva, Natalia V., Shvyreva, Uliana S., Nikulin, Alexey D., Tutukina, Maria N., Ozoline, Olga N.
• Format: Article
• Language: English
• Subjects: E. coli; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Escherichia coli Proteins - toxicity; Expression; ExuR; Gene Expression Regulation, Bacterial; LeuO; Operon; Purification; toxic » transcription factors; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism; Transcription Factors - toxicity; UxuR
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2019.05.001

Transcription factors play a crucial role in control of life of a bacterial cell, working as switchers to a different life style or pathogenicity. To reconstruct the network of regulatory events taking place in changing growth conditions, we need to know regulons of as many transcription factors as possible, and motifs recognized by them. Experimentally this can be attained via ChIP-seq in vivo, SELEX and DNAse I footprinting in vitro. All these approaches require large amounts of purified proteins. However, overproduction of transcription factors leading to their extensive binding to the regulatory elements on the DNA make them toxic to a bacterial cell thus significantly complicating production of a soluble protein. Here, on the example of three regulators from Escherichia coli, UxuR, ExuR, and LeuO, we show that stable production of toxic transcription factors in a soluble fraction can be significantly enhanced by holding the expression of a recombinant protein back at the early stages of bacterial growth. This can be achieved by cloning genes together with their regulatory regions containing repressor sites, with subsequent growth in a very rich media where activity of excessive regulators is not crucial, followed by induction with a very low concentration of an inducer. Schemes of further purification of these proteins were developed, and functional activity was confirmed. •Vectors were constructed and conditions were found to improve the yield of proteins, potentially toxic to a bacterial cell.•Using this approach, three transcription regulators from E. coli, ExuR, LeuO, and UxuR, were successfully overproduced.•Purification scheme to obtain significant amounts of electrophoretically homogenous UxuR and ExuR was developed.•The functionality of the purified proteins was confirmed by EMSA and ChIP experiments.