Zn finger proteins

Hi everyone,

In the last few months we have gotten a lot of orders for production/purification of Zinc finger proteins.  We are usually expressing in insect cells, and we can often get very nice expression, but we are unable to purify folded protein.  Either the majority of protein is insoluble or gets degraded during purification.  We have not seen much success with adding Zinc to the buffers.

Does anyone have any good tricks for Zn finger purification?  I have read that you should avoid His tags as both the His tag itself and the imidazole can chelate Zinc.  We also avoid adding DTT.

Thanks for any advice!
 

best,

Peggy

 

Posted on 20-Sep-2013 15:58 CEST

Hi Peggy,

Ni-resins can cause problems with Zn-finger domains, I guess especially if you have not all the Zn-binding clusters occupied by Zn. Ni2+ has been shown to bind and enhance oxidation of SH-groups.

Did you try adding Zn in the medium during growth instead of only in the purification buffers? Perhaps loading the Ni-resin with Zn instead of Ni-ions could help. Maybe you could use MBP as a purification tag, GST could be a problem with the high GSH conc. during elution which could weaken the Zn-binding.

Another cause of the low solubility might also be that the protein is bound to nucleic acid which then can end up in the insoluble fraction. High concentrations (1M NaCl) could help getting some soluble protein. This worked with a mRNA-binding protein in our lab (FMRP).

I hope that helps.

Hüseyin

Posted on 20-Sep-2013 16:21 CEST

Hi all,

we have a slighly different experience but i guess that this could be helpful in some sense:

For our project on Ciona intestinalis transcription factor we cloned the 426 DNA BD and tried to express/purify them all (in E.coli) with a histag. Out of 426 proteins, we could purify and characterize 266 with HTP SELEX mainly through a TRX fusion (see our MEthods paper in 2011). That was 77 % overall sucess on the non Zn finger DNABD and only 47 % on the Zn finger (around 220/426 Ciona DNA BD are Zn finger..).

People working on Zn Finger told us exactly what you said: Zn finger need Zn in the medium and in the buffer. They should be away from Histag. Because we are using ZYP5052 and because we kicked out the metal mix years ago we did regrow them in the presence of the metal mix. Because subcloning in Gateway takes only few days we subcloned all the failures in a StrepTRX vector and did the experiement again; we could recover less than 10% (don't remember really, the kind of things that you can not publish, I could dig but this is the order of success that we got...).

So our conclusion was : there is something else that is wrong here (folding, domain design...) and that was the end of this project...

So try the strep and the Zn all the way but be ready to try something else...

Renaud

Posted on 20-Sep-2013 16:25 CEST

Hi Hueseyin and Renaud,

Thanks for the tips.  We have not yet tried adding Zn to the media (I was assuming serum-free insect cell media must already have enough Zn, but maybe this is not right. . . ).  How much do you add?  We have also been thinking about Zn resin instead of Ni, just haven't gotten around to making the resin yet.

For one protein we did see slight improvement with MBP, though it was a His-MBP vector (construct design was not done by us!).

I also have the feeling that in at least some cases (already using strep tag, low temp expression) something else is going on.  Has anyone had any luck with detergents?  I think we will try the 1 M salt.  Also I think an earlier post in another thread from Mario mentioned using urea to get rid of bound DNA.  Any experience with that?

 

Thanks again!

best,

Peggy

Posted on 20-Sep-2013 18:06 CEST

Hi Peegy,

I do not have experience with Zn finger proteins, but I know that must grow in the presence of Zn in the medium (at least we have done it for Zn binding proteins in E.Coli, and avoid Ni columns and imidazole elution that can sequester your Zn from the protein.

Regarding urea, I use urea to eliminate RNA from RNA binding proteins with a very high affinity for RNA. I use 4M urea, but then I refold my partially denatured protein on-column. Large procedure, that can be use in cases like this of very high binding to nucleic acids (not necessarily your case)

We have very good experience using MBP sa  and the a tag in insect cells: 1) higher and more soluble expression, 2) you can easily purify your protein with Dextrin Sepharose™ High Performance from GE, and then get rid of the MBP with TEV protease

Posted on 21-Sep-2013 13:43 CEST

Hi Peggy,

One experience with a zinc finger protein and I did it with success with a strep tag.

Frederico

 

Posted on 23-Sep-2013 17:03 CEST

Dear Peggy,

probably too late for a reply. We have different experience with that: on the one hand we have succesfully purified Zn Finger proteins on Ni beads (His-Sumo tag) from E.coli. But we also had a Zn Metalloprotease, that was completely insoluble in E.coli. We purified tagless form inclusion bodies (which are replacing a tga anyway) and refolded active enzyme in a Zn containing buffer. Generally, we don't add Zn in the media, but in the first steps in protein purification, 100µM.

Best

Sabine

 

Posted on 29-Oct-2013 9:31 CET
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