Purification of the YihW Transcription Factor from the Probiotic E. coli Strain Nissle 1917 Using Affinity Chromatography on Spin Columns
Abstract
Due to their toxicity, bacterial transcription factors are difficult to be expressed, while their ability to form oligomers makes purification challenging. For each such protein, an individual approach is usually needed. Here, a vector for overproduction of the YihW (CsqR) from probiotic Escherichia coli Nissle 1917 (EcN) transcription factor was constructed, and optimal conditions for its synthesis in homologous expression system were selected: expression in BL21(DE3)-Codon Plus-RIL; induction with 50mM IPTG for 5 hours at 37°С. A rapid and effective purification scheme was developed, based on the affinity chromatography on spin columns with the Ni-NTA sorbent and combining native and denaturing conditions, followed with step dialysis. The developed approach allows obtaining homogenous, 95% pure, functionally active protein. Activity of the produced YihW was confirmed using complex formation with its DNA target and subsequent electrophoretic separation of the DNA-protein complexes in polyacrylamide gel.
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