GST Fusion Protein Purification Problems

06 June 2017 6 6K Report

Hey guys,

I'm having some difficulty purifying my 46 kD GST fusion protein, specifically, I'm having issues with proteolysis and purification. I appreciate all suggestions, however time and money are not on my side, so the simpler the fix or idea the better.

Induction:

To begin, I pick a colony from my colony purified GST fusion plate (transformed into BL21 cells about 3 months ago) and place it into a 20 mL O/N starter culture. Also, I'm wondering if the age of the E coli could have anything to do with proteolysis. The next day I add the starter culture to 180 mL of LB+ AMP and induce at OD600~.2 with 1mM IPTG, 37C for 5 hours. After induction I spin down my culture at 6000 x g 4C for 30 minutes.

lysis:

On day 3 I thaw my cell pellet on ice for about 15 minutes and resuspend my it in my lysis buffer 5mL/ cell pellet(50 mM tris pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM DTT, .1% triton X-100, .5M EDTA and 1 pierce protease inhibitor tablet per 10 mL lysis buffer all in PBS). I also add 1 mg/mL lysozyme and incubate 37C for 1 hr in a shaking incubator. I then sonicate my lysate on ice using a branson sonifier 250 (Output control: 6, Duty cycle %: 60%) for 15 pulses then off for 1 min ten times. Afterwards the sample becomes very viscous. I add DNAse 1 10U/mL and 1mM MgCl2 plus an additional 5 uL of 1M MgCl2 / mL cell lysate and let that sit on my bench at RT for 30 minutes with occasional shaking. After I spin down the lysates at 15,000 x g 4C for 30 min. I check the induction on a 4/14% SDS PAGE gel and see induction in supernatant (though it is mostly in the pellet and I have tried extensive denaturing and refolding to no avail).

GST-column purification

I thaw my 5 mL of supernatant on ice and prepare my column of 1 mL glutathione sepharose 4b beads and set up my column apparatus (wash with 10 column volumes PBS). Additionally, I add another of the same type of protease inhibitor tablets (1 tablet/ 10 mL). The apparatus is set up in a cold room (4C) and consists of the column and a peristaltic pump set to a flow rate of .5mL/min as well as the collection vessels set up in an ice chest. I load my supernatant onto the column and collect the flow through. I then wash the column 5 times with 3 mL PBS then elute 4 times with elution buffer (50 mM tris pH 8, 10 mM reduced glutathione in water).

I was concerned I may have been overloading the supernatant on the column so I tried different dilutions in water.

Lanes: 100% Sup, 50% Sup, 25% Sup, FT, W1, W2, W3, W4, W5, E1, E2, E3, E4.

Concerns:

My supernatant lanes are very smeary and I'm not sure why, but this is a relatively recent occurrence whereas the pellet (not shown) has clean bands. Possible proteolysis of protein since I see the bottom group of thick bands in elution. Continual loss of proteins in washes.

My main questions are if my lysis conditions, specifically sonication conditions is too harsh, why are my supernatant lanes just recently becoming smeary (despite not changing induction parameters or volume loaded), as well as any ways to further reduce the proliferation of the bottom possible proteolysis bands.

Thanks for taking the time to read my question!

Benjamin Bauer

Thanks for your response Yanmei,

do you think I should be concerned about the smear in my supernatant lanes? The supernatants had distinct bands up until at least the end of April and have only recently been occurring. Is it possible that there is something wrong with the colonies ( I haven't refreshed them for about 3 months now).

Also what kind of recommendations do you have to improve my wash? More of them? How does decreasing the amount of beads help with my purification? Do you think the bottom three thick bands are proteolysis fragments?

thanks!!

Nicholas Spellmon

Hi Benjamin,

I have several suggestions you could try for promoting solubility and purity of your protein. In the future, load less protein on your SDS-PAGE next time so it doesn’t smear!

Solubility. (1) Try growing cells at a lower temperature (15-20°C) and grow overnight (16-20hrs). This should allow more time for your recombinant protein to fold properly and stay in the supernatant after lysis. (2) Try a french press if available. Sonication can damage the fold of your target protein, and french press is usually less damaging. If the supernatant is viscous after sonication, you should include DNase. Adding DNase (or lysozyme) shouldn’t effect purity in this type of purification.

Purity. (1) Try a low salt buffer wash [100mM NaCl] followed by a high salt buffer wash [300-500mM NaCl]. This can help eliminate non-specific proteins binding to the resin or your protein. (2) Are you using BL21 or BL21(DE3) cells?

Hope this helps!

Micah Jered Ferrell

Not knowing what your MW markers are I am taking a guess here, but it looks like you may have incomplete expression products. Assuming the highest MW band in you elution lanes is the desired product, your smaller co-eluting bands could be indicative of GST-tagged fusions where the full-length gene did not express. This could be due to plasmid instability in the host (part of the insert was lost) or possibly due to expressing the protein too rapidly (ribosome is falling off before the end).

I would recommend re-transforming into expression host and seeing if that clears it up. I frequently make glycerol stocks of plasmid in expression host- I don't recommend it for plasmid propagation but it ensures more stability than leaving things on a pate. Just scrape the top of the glycerol-culture slush with a tip and inoculate a starter culture.

Also, consider expressing longer at lower temperatures. If you are using the the right "BL21" cells you may also be able to try altering concentrations of IPTG (i.e the "Tuner" series from Novagen) to slow down expression and decrease the chance of things going awry.

Another pro-tip, look up auto-induction media (Studier, 2005 & 2014), it will change your life. It is inexpensive, fairly easy to make and amenable to long low-temp expressions. Trace metals (the only part that is hard to make) can be purchased from Teknova and will last forever.

Benjamin Bauer

thank you all for your helpful advice.

Yanmei- I will try a fresh colony.

Nicholas- The strange thing about the smearing is that it has only recently developed. As of a month ago, I had clean bands and I haven't changed my protocol for induction, lysis or SDS PAGE (including keeping the volume constant). Additionally, I only see smearing in the supernatant, and not the pellet (not shown). The first 3 lanes are the supernatant, 1/2 supernatant diluted with water, then 1/4 supernatant diluted in water. It looks clearest in this third lane, however it is still not as clear as it used to be only a month ago. I'm using the regular BL21 cells, but I also am using pierce protease inhibitor cocktail that has an aspartic acid protease inhibitor in it.

Micah- I'm using precision plus protein standards all blue so the MW ladder is (in kD): 250, 150, 100, 75, 50, 37, 25, 20, 15, 10 and my protein is at 46 kD so on the gel in the first elution lane it's the second thick band from the top and is present in all 4 elutions. If re-expressing my plasmid doesn't help I'll start thinking about solubility. Thanks for the recommendations.

Ankita Dutta

Please use a fresh colony for protein expression in bl21plyss strain. Also, try and not expose your plate to 4c. Induction with IPTG is recommended at 0.5 to 0.6 OD... you cany optimize the induction by growing cells at 37 C with varying concentrations of inducer 0.05 to 1mM for 3 to 4 hrs or 30C for 6 hrs using a same inducer concentration...You can preserve your pellet for uptown a month in -80...Thaw your pellet on ice and add your resuspension buffer with the protease cocktail to completely dissolve the pellets by intermittent vortexing... You can add lysozyme final concentration 0.2mg/ml even in thawed inllet kept in ice for not more than 20 min n sonicate... You could increase your binding time to beads to about 4 hrs by twirling the supernatant and the beads and then do gradual washing with PBS about 20 column volumes and then with 5mM glutathione followed by increasing concentrations of glutathione... the protease inhibitors are less likely the problem... All the best!!!

Benjamin Bauer

Hey guys so I retransformed my plasmid and it looks a lot cleaner. I'm still having trouble purifying the fusion protein and have started a new discussion here:

https://www.researchgate.net/post/Multiple_bands_in_GST_fusion_protein_purification

thanks!

Multiple bands in GST fusion protein purification

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