Hi all,

We have been trying to produce 3C (pET/3C) we got from Arie Geerlof using the protocol available at his lab website. For some reason we have been obtaining dirty preps directly from the IMAC purification, and after a SEC the yields are too low (in the order of 0.3 mg/mL - by OD280 measured by Nanodrop), despite the qualitative (by-eye) SDS-PAGE band quantitation seems better than that.

Does anyone had similar problems?

All the best,

Pedro

Hi Pedro,

Firstly-for 3C protease it is essential to extract in the presence of detergent or the yields will be really low. We use 0.2% Tween 20 with the cell disruptor and I think Arie's original protocol used NP40 or Triton. Do you have detergent ?

After that what are your culture conditions nd IMAC buffers?

Nick

Dear Nick,

You nailed it...

We were not adding the detergent to the extraction buffer, and that was the only difference with Arie's protocol.

Thanks for the tip!

Pedro

Hi Pedro,

actually we use a lysis buffer without detergent (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM imidazole) and did not observe any problems so far. Expression levels are quite high, we obtain roughly 30 mg of pure His3C or more per litre culture. Lysis is done by sonication.

We don't do SEC as the 2nd step but one anion exchange step with Q-sepharose where His3C passes through nicely and most contaminants are bound to the resin. This is much more convenient and faster than SEC with such high volumes of protein.

This works also nicely with HisTEV protease by the way.

Let me know if you need more details.

Best regards

Hüseyin

Hi Pedro & Nick,

I wonder if the lack of detergent alone really is the one detrimental factor causing all your problems.

We never use any detergent in our 3C preps and tend to get at least ~600-800mg from a 2L culture (auto-inductio, RIL).

Have to admit though that we are not using SEC, but dialysis, as the purity after IMAC is perfectly fine for our purpose (>90%).

@all: are we completely alone in this or is anybody else NOT using detergents

Cheers,

Tim

Hi All,

Perhaps it is a combination of lysis method and salt concentration?

Certainly in 500mMNaCl if we lyse with a cell disruptor we extract only a few percent of the 3C in the absence of detergent (or at least when I tried it last!) so we have continued lysis with the detergent. 

Lower salt concentrations improving extraction also makes some sense as it may remove the need for the addition of detergent.

How are you lysing the cells in your labs Tim and Hüseyin?

Nick

Hi all,

Could you share more details of your 3C prep protocols (in particular Hüseyin and Tim)? 

We are expressing the pET/3C construct in Rosetta cells with autoinduction ZYM5052 media. After growing cells at 37ºC up to OD600 of 1.0 we decrease temp to 20 ºC and let them grow for 16h (do you let them grow for longer periods?).

Cells are resuspended in 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole, 10% glycerol and lysed through a cell disruptor (Avestin C3). After centrifugation the sup. is loaded into 5 mL HisTrap HP (GE) and eluted with 150 mM imidazole (As Arie's protocol suggests).  I see that Tim's protocol uses 500 mM imidazole, how much imidazole are you using, Hüseyin? - That may explain our low yields...

The SEC was an attempt to clean the prep, as the eluted protein from HisTrap was a little dirty, but the protein ammount was not good - we have recovered around 1.2 mg total protein...

Another question for Hüseyin: After IMAC and prior to negative Q sepharose, you should desalt/dialyse your protein as (I believe) you have NaCl in your elution buffer, right?

Thank you all for yor inputs,

Pedro

Hi Pedro,

please check the pdf that I uploaded to the "files" section. It contains the protocol including gel pictures of our last purification.

Best regards

Hüseyin

Could anyone update me on sequence information for recombinant 3C proteases. Any mutations that have significantly improved cleavage or stability? I wasn't able to find a reported systematic analysis.

Hi Gro,

I guess you mean mutations in the 3C protease not in the recognition sequence. I haven't heard or read about mutations improving the activity and/or stability but one deamidation reaction in an Asn side chain seems to decrease the activity significantly, as described here http://www.ncbi.nlm.nih.gov/pubmed/10224078, which leads to this paper (http://www.sciencedirect.com/science/article/pii/S0042682200907608) citing the first one and describing an increase of 3C activity in 0.8M sodium sulfate.

Maybe activity differences are caused by this deamidation and one should be careful with storage or cleavage conditions that could enhance the deamidation reaction.

I'm not sure if that was helpful for your problem.

Best regards

Hüseyin