I constructed two plasmids for DnaA expression: one with an N-terminal His-tag and the other with a C-terminal His-tag, and neither tag was cleaved during purification.The purification buffer used was 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 10% glycerol, with storage at -80°C. For activity testing, I performed electrophoretic mobility shift assays (EMSA) using two distinct DNA substrates:
On a 1% TAE gel, I observed migration retardation only for the chimeric plasmid, whereas the oriC fragment showed no detectable binding. I am uncertain whether the retained His-tag interferes with enzymatic activity. Additionally, many published protocols for DnaA purification utilize Hepes/KOH (pH 7.6) instead of Tris-HCl, raising concerns about potential buffer-specific effects on activity.
Furthermore, literature methods often employ potassium glutamate at varying concentrations to prevent DnaA aggregation. However, in my experiments, when using 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 10% glycerol, overnight storage at 4°C caused significant protein precipitation. Adjusting the buffer pH to 7.5 (based on DnaA’s isoelectric point) resolved aggregation issues. This prompts the question: Does salt type (e.g., NaCl vs. potassium glutamate) also influence enzymatic activity?
Key Questions:
Try reducing the NaCl concentration to 150 mM or less. 500 mM is much higher than physiological ionic strength. The interaction with DNA probably involves some electrostatic interactions, which are going to be diminished at high ionic strength.
Thank you for your guidance, Professor. I will proceed with the following experiments:
1、Parallel Purification & EMSA Validation: Simultaneously purify both C-terminal tagged and non-tagged DnaA proteins. Reduce the salt concentration in the purification buffer to 100 mM NaCl while keeping other conditions unchanged. Repeat EMSA assays post-purification using two distinct substrates: oriC DNA fragment and Chimeric plasmid containing oriC fragment.
2、Substrate Topology Investigation: Explore whether the topological state of DNA substrates affects DnaA binding by:Linearizing plasmid DNA via restriction enzyme digestion for EMSA testing.Compare binding affinity between supercoiled (intact) and linearized plasmid substrates. Hypothesis Testing: If DnaA binding disappears with linearized DNA, this would suggest: Conformational dependence: Supercoiled DNA topology enhances DnaA binding efficiency. Affinity threshold: Linear DNA may require significantly higher protein concentrations to achieve detectable binding.@Adam B Shapiro
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