Hello, everybody.
I am looking to add a DNAse treatment step to my bacterial lysis (gram-negative) procedure for crude enzyme extraction because my lysate always ends up being too viscous. We only have Zymo Research DNAse I in the lab and its information sheet says not to "avoid phosphate buffer and calcium chelators". However, I am using a chemical lysis procedure using Promega Cell Culture Lysis Reagent (following their bacterial lysis protocol) since we do not have a sonicator available. According to their information sheet, CCLR has 25mM Tris-phosphate (pH 7.8) and 2mM 1,2-diaminocyclohexane-N,N,N´,N´-tetraacetic acid. Does anyone know if adding DNAse I to my lysate will still work?
I am not sure of the composition of Zymo's DNA digestion buffer, but I was thinking of supplementing divalent ions to the lysate to counter the 2mM chelating agent in the lysis buffer. However, I am not too sure how to circumvent the phosphate problem. I am not sure how phosphate affects DNAse activity exactly. On the other hand, Thermofisher has a protocol for removing DNA from protein extracts (extracted using lysis reagent in phosphate buffer) using DNAse I. Will the phosphate component of my buffer significantly affect the activity of the DNAse?
Any insight on this matter will be greatly appreciated. Tips on how to solve the viscosity problem altogether are also greatly appreciated.
Thanks
I am not aware of phosphate inhibiting DNaseI so I would just go ahead and supplement with cations above the concentration of the chelator.
Hello Christiana Lagudas
The following are the conditions when DNase I activity can be affected.
1. DNase I activity is influenced by divalent ions. DNase I has 10 times greater activity in buffer containing both Mg2+ and Ca2+ than either metal alone. Calcium is required to maintain structure and activity of DNase I. Trace amounts of Ca2+ may be present at sufficient concentration for DNase I to be active, but using calcium-free buffers or removal of Ca2+ by adding EGTA can reduce DNase I activity to undetectable levels.
2. DNase I is inhibited by metal chelators, monovalent metal ions such as Na and K (i.e., ≥ 100mM NaCl), SDS even at concentrations below 0.1%, reducing agents and ionic strength above 50-100mM.
3. DNase I is inactivated by heating to 65°C for 10 minutes in the presence of EGTA or EDTA.
4. DNase I is sensitive to physical denaturation. So, mix gently by inverting tube. Do not vortex.
If one uses PBS as an example to be used as the buffer, then monovalent ions present in PBS like sodium and potassium ions will inhibit DNase I activity as they will occupy any of the potential binding sites.
The contents of (1X) PBS are as follows.
137mM NaCl,
2.7mM KCl,
10mM Na2HPO4, and
1.8mM KH2PO4
However, the inhibition of DNase I can be reversed by adding divalent ions. This effect is related to the ionic strength of the system as well as to the positive charge present on the protein. I have not come across anything about phosphate on DNase I activity.
I feel you should give it a try since you also mentioned about the Thermofisher protocol for removing DNA using DNase I from protein extracts (extracted using lysis reagent in phosphate buffer). But do supplement divalent ions to the lysate.
There are a few articles which you may want to refer for more information.
Article Deoxyribonucleic Acid Nucleases
https://www.jbc.org/article/S0021-9258(18)91214-7/pdf
Article CRYSTALLINE DESOXYRIBONUCLEASE
https://www.sciencedirect.com/science/article/abs/pii/S0223523414006473#:~:text=An%20effective%20and%20simple%20method,sodium%20citrate%20(pH%207.6).
Best.