I'm working on the purification of a few different his tagged proteins and while some have turned out good, other preps present this strong yellow colour after purification. I've expressed in a few different E. coli cell lines and sometimes I get the yellow colour and other times I don't, so it doesn't seem to be related to that. I lyse the cells with a little lysozyme and two cycles of 7 min of sonication, after what I spin down and filter the SN. The purification method is a simple His trap followed by size exclusion chromatography, using buffers like 25mM Tris ph 7.5, 200mM NaCl and added imidazole for the elution buffer. When I run these samples on a SDS-Page I get this very strong and rather pure protein around the size of 60KDa, that is not my protein of interest. Has anyone ever seen anything like this?
Dear Ana
is you protein contain free cysteines and are you suing NI-NTA for IMAC?
Because in the past i observe orange elution when for Sco2 proteins, which is a copper binding protein which bind copper through an C-X-X-X-C conserved motif, i performed an IMAC using the Nickel instead Zinc as metal in the IMAC and the protein elute orange because it was able to extract nickel from the coloumn.
In those case, to produce the not coloured apo form, the addition and incubation with an excess of EDTA and removal of Nickel-EDTA complex by desalting was enough to solve the problem.
Ciao
Manuele
What is the size of your proteins of interest? SEC isn't enough to separate these proteins from the 60-kDa contaminant? You can always try to change your buffer conditions, or you may run a mass-spec to identify the contaminant, maybe it will help you to resolve the problem (maybe by changing washes, or changing resin etc.)
And with what concentration of imidazole does it elute? That would be important to know wheather it interacts with the resin or not.
A bright yellow color may arise from flavin binding proteins, but you cannot be sure without running a mass-spec.
Hi Jorgac, thanks for your answer! They all have different sizes so SEC potentially separate the smaller ones from this 60KDa one. The problem is that because the cells are overexpressing this 60KDa protein they’re not expressing enough of my protein of interest. I had already thought about doing mass spec so thanks for the suggestion!
Hello Manuele, thanks for answering. what you said makes sense but the problem here is that in these yellow preps I can’t see the protein I’m trying to purify but instead a 60KDa one that’s being overexpressed. This has also happened in other colleagues preps and we can always see the same pattern on the sds page, a smaller fainter barn followed by a strong one at around 60KDa.
Hi Ana, this is a curious problem, which i think prompts a couple of questions worth looking at. Generally i would wager to say that a highly abundant contaminant of 60 kDa that is yellow is not typical of common E. coli expression strains (this is my experience at least). The fact that it occurs across strains could suggest some kind of contamination - whether it be in your cultures or your plasmid prep is contaminated with another vector. Since the contaminant seems to bind your Ni-NTA nicely my immediate suspicion would be contamination with a strain or vector harboring another recombinant his-tagged protein.
Firstly, i would check very carefully what you are doing to generate your overexpression strains. Which vector are you transforming with, did you prepare it yourself and is it sequenced so you know exactly what gene is being induced in your experiments? Additionally, it may be worth testing whether this mystery 60 kDa yellow protein is dependent on IPTG induction or not. A quick small scale test experiment doing his-tag purification from either induced or non-induced culture should tell you this. Alternatively you might be able to tell whether the protein is present in lysates without purification (it looks like it is quite abundant). In terms of finding the source of potential contamination that can be a tricky issue, and it may be better to purchase fresh commercial expression cells and keeping them strictly separated from other used strains in your lab.
Second, to get you a bit closer to what you are actually purifying, how about recording a UV-Vis spectrum of your purified fraction with the 60 kDa protein. In some cases (e.g. flavins) this can yield a quite characteristic spectrum. If you suspect flavoprotein, you can do a number of additional things, e.g. look at fluorescence (450ex, 525em) before and after extraction of FMN/denaturation of protein (e.g. boiling for 5 min - beware though, some flavoproteins do not denature under these conditions). Extracting the FMN also allows you to perform HPLC or LC-MS to identify the cofactor (whether flavin or not), as suggested by Jorgac.
Hope this helps, best regards,
Silas
Do you see a lot of your protein of interest in the insoluble fraction? In some cases when the cells are stressed with a heavy load of overexpression they will respond with expressing various chaperones. your 60 kDa protein may be a chaperone.
Bo Pontoppidan i think you were right! I did mass spec on this sample and it shows a 57KDa protein which when compared to native E. Coli proteins makes me suspect is Hsp60 which is commonly co-purified in IMAC. A WB on the cell pellet showed a massive amount of insoluble protein. Thanks for you great answer!!!
- Badges
- Science topic