Overproduction and purification of the Escherichia coli transcription factors "toxic" to a bacterial cell.

Autor: Bessonova TA; Institute of Cell Biophysics of RAS, Pushchino, Moscow region, 142290, Russia; Institute of Protein Research RAS, Pushchino, Moscow region, 142290, Russia; Lomonosov Moscow State University, Moscow, 119991, Russia. Electronic address: tatianabessonova66@gmail.com., Lekontseva NV; Institute of Protein Research RAS, Pushchino, Moscow region, 142290, Russia., Shvyreva US; Institute of Cell Biophysics of RAS, Pushchino, Moscow region, 142290, Russia; Institute of Protein Research RAS, Pushchino, Moscow region, 142290, Russia., Nikulin AD; Institute of Protein Research RAS, Pushchino, Moscow region, 142290, Russia., Tutukina MN; Institute of Cell Biophysics of RAS, Pushchino, Moscow region, 142290, Russia; Kharkevich Institute for Information Transmission Problems RAS, Moscow, 127051, Russia., Ozoline ON; Institute of Cell Biophysics of RAS, Pushchino, Moscow region, 142290, Russia.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2019 Sep; Vol. 161, pp. 70-77. Date of Electronic Publication: 2019 May 01.
DOI: 10.1016/j.pep.2019.05.001
Abstrakt: 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.
(Copyright © 2019 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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