Hello everyone,
I seek a little assistance with my stubbornly insoluble protein. I express my protein in E.coli BL21 (DE3) cells with a suitable tag for solubilization. To be precise, my conditions are: LB broth, induction at 0.4 OD, 0.42mM IPTG, 20oC for 20 hours after induction.
Though, I get 70% soluble protein when I maintain a lower culture volume (~10-15ml) and have dissolved the pellet in almost equal amount of buffer (Tris HCl pH 8, NaCl somehow interferes with my protein so I do not add) for lysis through sonication but the same does not occur by increasing the culture volume (~25ml). I had even tried to use 200µg/ml Lysozyme (37oC for 30mins) prior sonication but that did not work too.
To this end, I would like to know whether anyone of you had ever faced such similar complications during protein expression. If yes- kindly help me out!
There is a lot of misinformation in your concern. Is the protein being expressed as a soluble entity or as inclusion bodies? Do you have cell breakage problems or not? What really happens when you change scale in terms of expression form. I have no idea how you were able to "dissolve" a pellet of whatever using Tris.
So, is the problem with solubility when you increase the volume or with protein extraction (you mentioned you added lysozyme, so I guess you believe you just couldn't break up the cells)? As Omar stated, it would first be important to know whether your protein is soluble or not in your 25 ml batch. Simply run an SDS-PAGE of the soluble and insoluble fraction and let us know.
Difference in recombinant protein solubility when cells are grown in different volumes of medium (we are talking here about 15 vs 25 ml, not 15 ml vs. 2 l) would not be expected if you use the same growth conditions (medium, flask, induction time, rpm).
In your 15 ml batch I guess you sonicated cell pellets in 15 ml Tris, then took the supernatant after centrifugation and your protein was present there (as judged from SDS-PAGE). Is this so? If you did the same with the 25 ml batch, you should see the protein in the supernatant as well, at least a comparable amount (of course assuming that cell growth was similar and that you used essentially the same extraction/analysis procedure). If this was not the case, let us know and we will think about your options. I tend to believe it is more a technical issue than something with your cells or protein. Be aware that sometimes cells loose the ability to express your protein after a while, so you might also check if you can still get your protein soluble in a 10-15 ml batch. If not, retransform and use fresh transformants for induction.
Thank you very much for the suggestions.
To start with, the protein basically is not a soluble one. However, I have attached a suitable tag to solubilize it (which worked to some extent). I have hereby attached a SDS PAGE image of 5ml culture volume (added 5ml Tris HCl buffer to the pellet for sonication) which solubilized the protein quite well. To this end, when I tried to scale up the protein to 25ml I got almost nil fraction into the soluble form (plus sonicating 25ml was very tedious).
All the conditions were same for both of them. I checked the final OD which was 2 for both.
Can scaling up lead to
PS: Does a little fluctuation in buffer affect the lysis (like pH 8 to 7.4) ?
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Let's first go through your 5 ml experiment and Pic1. So, lane 2 represents supernatant after sonication and centrifugation of the lysate. There is obviously quite a lot of solube protein. Is lane 3 representing cell pellet (cells before sonication) or is it pellet after centrifugation of cell lysates (after sonication)?
Please include a legend for Pic. 2 (lane-to-lane). There is a lot of protein in lane 4 (which I assume represents total cell lysate). This band should appear again in one of the fractions after sonication/centrifugation. My guess is that you did not analyze the insoluble fraction, but maybe I am wrong. Insoluble fractions should always be analyzed in this type of experiments.
A change of pH from 7.4 to 8.0 could be important if you are somewhere close to the pI value of your protein. Otherwise, a shift towards slightly alkaline shouldn't really matter for most of the proteins.
When you say it was tedious to sonicate the 25 ml lysate, what exactly do you mean? Did you observe an initial increase and then decrease of viscosity? Could you easily separate the two fractions after sonication?
Thanks Marco.
For picture 2, the blue arrow indicated my protein in soluble fraction. Lane 8 is the pellet.
When I sonicate for 5ml culture, it becomes almost transparent. However, for 25ml culture volume I divide them in 8 fractions (each 3ml) and sonicate. Thus, I mentioned it to be tedious.
Regarding, pH change, pI of my protein is 5.9 and I had achieved better lysis in pH 8 Tris HCl . I am asking whether it might be an issue if pH drops from 8 to 7.4~5 ?
One more thing that I have discovered recently is that my protein's estimated half life is 10 hours in E.coli (invivo). After induction I incubate for 20 hours at 20oC. Can this play any role in trapping my protein into the insoluble fraction ?
Dear Bapi,
Kindly let me know, which vector you have used for the expression and kindly optimize your buffer condition also the lysis buffer condition need to be checked. For soluble protein expression try to use mbp tag vector.
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