Hello,
There is lacI protein which is binding to the expressed recombinant protein after purification using Protino Ni-TED 2000 packed columns. I have tried gel filtration to separate the proteins and still they are co-purified! I have tried different washing strategies such as (ATP, MgCl2, and UREA) and still not helping. So, I decided to use MONOQ column or ion exchange chromatography but the problem is different this time, I cannot elute any protein from the column! I used pH buffer of 8.5 because the PI of my protein is 7.5. I even tried high salt concentration up to 1.5 M NaCl but still the protein cannot be eluted! Is there any body could help with any advice? or somebody faced the same problem ?!
Note: I get to know that the binding protein is lacI after I did proteomics analysis.
Dear Nadia,
Dear Nadia,
your question is difficult to answer without knowing your target protein.
But maybe the binding sites of LacI for the lac operators/lactose are responsible for your problem.
This may be the case if your protein of interest and LacI bind to a piece of DNA you are copurifying. You can add DNase to prevent this (or ensure its activity if you already use it - Mg and Ca salts as cofactors often increase the activity of DNase preparations).
If you are sure the extract is DNA-free, deliberately adding DNA containing a lac operon may induce a conformational change to LacI and weaken the interaction with your protein of interest.
DNA-biding of LacI is achieved through basic residues - mainly arginine - which may bind to anionic sites in your protein of interest (such a site might be responsible for the strong binding on the anion exchange column). Adding Arg to your buffer may saturate these sites and prevent such interactions. Arg also has a stabilizing effect on some proteins.
If your protein of interest is glycosylated LacI might attach there. An excess of lactose in your buffer could prevent that. Lactose also induces a conformational change in LacI that could help you get rid of it. The addition of sugars can benefit protein stability as well.
I hope this gives you some ideas. Best wishes for your purification!
Kind regards,
Lorin
Dear Lorin,
My proteins of interest (4 enzymes) are belonging to NADH-flavin oxidoreductases, old yellow enzyme family. The problem of lacI binding is happening with the 4 enzymes. You can see the attached file for the SDS-PAGE image. I also believe that the lac operators/lactose is responsible for this problem. I will give a try to the DNase, do you have a detailed protocol for using DNase? I actually have tried to express the 4 enzymes from Strep-Tag but they were produced as inclusion bodies with no activity. The expression system which gave me nice activity is the one which makes this binding problem and it is called pt7-flag-mat-tag-1.
The gel filtration technique is separated on the basis of the partial weight, and you must first determine the partial weight, and then choose the appropriate Sephadex, that is, we must choose the size of the Sephadex particles suitable for the partial weight of the enzyme to be separated. And you know that if the enzyme consists of several monomers, then one package appears in the gel filtration technique, but the anion exchange technique shows us the monomers that make up the enzyme
Dear Nadia,
if you are buying DNase you should follow the vendor's protocol. Otherwise, you can try the following conditions:
Buffer Components
10 mM Tris-HCl pH 7.5
2.5 mM MgCl2
0.5 mM CaCl2
Reaction time
~30 min at 37 °C
Notes
Na+ and K+ should be below 100 mM.
Avoid reducing agents (DTT, 2-Mercaptoethanol), chelators (EDTA, EGTA), transition metal salts (e.g. Zn2+) or surfactants (SDS) in your buffer as these may inhibit DNase activity.
Depending on the amount and activity of the DNase you're adding, reaction time may be shorter/longer.
You should notice a marked decrease in viscosity of the cell lysate as the DNA is degraded.
Kind regards,
Lorin
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