his-tag protein pure but degradated during growth - his-tag protein pure but degradated during growth (Apr/28/2008 )

Hi everybody! I'm working with a 74KDa his-tag protein cloned in BL21 (DE3). I've optimized the conditions for an IMAC cromatography with Nichel sepharose resin in denaturing conditions (6M urea). On a SDS-PAGE different shorter fragments are visible together with the full-lenght protein (good yield), but I've confirmed by western blotting that they derived from the target protein degradation. I always work at 4°C and as fast as possible, and I add the inhibitors cocktail (EDTA free) to the lysis buffer. I've tried to reduce the induction time and temperature but I didn't see any improvement, so I believe that the proteolysis happens during the IPTG induction time (1 or 2 hours). Do you think it's possible? If yes, how can I reduce it? And I can I purify my protein from its fragments? I've already tried with a gel filtration in native conditions but it didn't work... Maybe an ionic exchange? Or hydrophobic interaction? Thanks in advance!

---

If your protein is unstable you might have to have a very short induction. Grow in a rich medium until your OD is quite high then induce for a short time. You can use glycerol as the carbon source.

Alternately, you might need to look at cell-free expression, in case your protein is being chewed up by the cell's proteolytic machinery.

---

What surprised me is that the proteases are still active after your elution

Did you elute your protein under denaturing conditions or Native ?

Have you tried using GnHCl instead of the Urea ?

---

I tried short induction time as 1 hour. Do you think I have to induce for still shorter time?
I'm afraid that cell-free expression allows the recovery of max 1mg of target protein, isn't it? I need to scale up the production after the optimization tests...
Probably I need an eukariotic expression system...
thank you

---

Why do you think that the proteases are still active after the eluition? I think they degrade my protein during the cell growth and/or the induction time.
I elute the protein in denaturing conditions, with urea 6M. Why GnHCl? I'm quite pleased with my purification protocol, but I need to eliminate degradation during cell growth, maybe adding another cromatographic step or... what?
Thank you!

---

Well i'm just trying to guess Maybe you should do a simple test to know where the degradation might occur !

1/ Do the induction of let's say 50ml of culture let it grow at 25°C and induce when you reach the right OD (0,6 to 1 OD unit)
After induction collect 1ml of teh culture right away and collect 1ml each 30mn uup to 4hours
immediately after collect pellet the bacterias and resuspend in SDS sample buffer

Analyse on the gel and look whether you see the protein and whether you see appearing the degradation during the time !

if your protein is produced in too low amount you can try to do a quick purification in batch with your nickel agarose and do the same analysis

GnHcl beeing a stronger denaturant it might block protease in a better manner than Urea no ?

---

Why do you think the protein is degraded during expression? If that were so, I doubt you'd see anything at the end. What protease inhibitors are you using? Are they working properly? Try adding some to a known protein and treating it with trypsin or some other protease.

Do you have a gel image you could show us of the amount of degradation? Which end is the His tag?

If you really want to separate full-length from breakdown, IEC might work, but it's hard to predict. Have you tried native PAGE to see if there are any differences?

HIC also might work.

I have a sneaking suspicion that the problem lies with the protease inhibitors.

---

I'm not sure but I thought that proteolysis happened during expression because I've tried to change induction time (from overnight to just 1 hour) and lysis method, I've tried to thaw or not the pellet before lysis, and to add or not protease inhibitors cocktail to the lysis buffer: nothing changed! I thought that the proteolysis happened when there were no proteases inhibitors, but NOW I think you could be right, maybe the "Complete Protease Inhibitor Cocktail Tablets EDTA-free" doesn't work well in my condition or something like this... May the protein degradation be completely act by the metalloproteases, due to the absence of EDTA? Do you know any other method that can inhibit metalloproteases but that allows the use of IMAC?

Just one more (maybe stupid) question: I still use protease inhibitors cocktail even if I directly resuspend the pellet in a denaturing lysis buffer containig urea 6M... Does it still work in denaturing conditions? (proteases should be denatured too... why they degrade my protein???)

---

Just for information: what exactly is your protocol?

---

BL21 recombinant cells are grown util a A₆₀₀=0.8-1.0 at 30°C, then the culture is shifted at 20°C and induced with IPTG 1mM for 1 hour. Cells from 1 liter of culture are collected and directly resuspended in 50ml of 20mM sodium phosphate buffer pH 7.4, 0.5M NaCl₂, UREA 6M, DTT 3mM, Imidazole 30mM and Protease EDTA-free inhibitor cocktail 1X (1 tablet). Cells are disrupted by sonication. After centrifugation, the lisate is loaded onto a nichel-sepharose column equilibrated with ther lysis buffer (without inhibitor cocktail). After washing with the same buffer, the protein is eluted with 250mM imidazole added to the same buffer. What do you think about it?

---