A simplified method for purification of recombinant soluble DnaA proteins

Protein Expression and Purification
Anna Zawilak-PawlikJolanta Zakrzewska-Czerwińska

Abstract

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.

References

Apr 1, 1997·Journal of Bacteriology·J MajkaJ Zakrzewska-Czerwińska
Sep 12, 1997·The Journal of Biological Chemistry·M D Sutton, J M Kaguni
Sep 25, 1999·Protein Expression and Purification·Z Li, E Crooke
Nov 26, 1999·Biochimie·W MesserJ Zakrzewska-Czerwinska
Apr 25, 2000·Journal of Molecular Biology·D JakimowiczJ Zakrzewska-Czerwińska
Mar 27, 2001·EMBO Reports·E BoyeK Skarstad
Jul 27, 2002·Annual Review of Microbiology·Gregory T Marczynski, Lucy Shapiro
Jan 14, 2003·EMBO Reports·Igor Konieczny
Dec 4, 2003·Journal of Molecular Biology·Anna ZawilakJolanta Zakrzewska-Czerwińska
Jan 10, 2004·Methods in Enzymology·Walter Messer, Christoph Weigel
May 13, 2004·The Biochemical Journal·Anna ZawilakJolanta Zakrzewska-Czerwińska
Jul 20, 2004·Nucleic Acids Research·Pawel MackiewiczStanislaw Cebrat
Aug 25, 2004·The Journal of Biological Chemistry·Takuma IshidaTsutomu Katayama
Feb 3, 2005·Molecular Microbiology·Alan C Leonard, Julia E Grimwade
Mar 26, 2005·The Biochemical Journal·Anna Zawilak-PawlikJolanta Zakrzewska-Czerwińska

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Citations

Jul 25, 2007·Molecular Microbiology·Anna Zawilak-PawlikAgnés Labigne
Aug 8, 2015·Cell Stress & Chaperones·Anna M GrudniakKrystyna I Wolska
Dec 15, 2016·Frontiers in Microbiology·Łukasz MakowskiJolanta Zakrzewska-Czerwińska
Oct 9, 2019·Microbiology·Małgorzata PłachetkaJolanta Zakrzewska-Czerwinska
Nov 13, 2019·Journal of Bacteriology·Tomasz ŁebkowskiJolanta Zakrzewska-Czerwińska
Jul 28, 2018·Frontiers in Microbiology·Pawel JaworskiAnna Zawilak-Pawlik

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