A simplified method for purification of recombinant soluble DnaA proteins
| Autor: | Jolanta Zakrzewska-Czerwińska, Anna Zawilak-Pawlik, Agnieszka Kois |
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| Rok vydání: | 2006 |
| Předmět: |
genetic processes
Streptomyces coelicolor Buffers Chromatography Affinity law.invention Mycobacterium tuberculosis Bacterial Proteins Affinity chromatography law Protein purification Escherichia coli Protein precipitation Histidine Helicobacter pylori biology biology.organism_classification Recombinant Proteins DnaA DNA-Binding Proteins Solubility Biochemistry Ammonium Sulfate health occupations Recombinant DNA bacteria Bacteria Biotechnology |
| Zdroj: | Protein Expression and Purification. 48:126-133 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2006.01.010 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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