A simplified method for purification of recombinant soluble DnaA proteins.
An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.
Zawilak-Pawlik
Anna M
AM
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Weigla 12, 54-114 Wroclaw, Poland. azawilak@pasteur.fr
Kois
Agnieszka
A
Zakrzewska-Czerwinska
Jolanta
J
D000645:Ammonium Sulfate / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002021:Buffers; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011994:Recombinant Proteins; D006639:Histidine; D000645:Ammonium Sulfate