Dear Colleagues

we obtained the Addgene clone and want to purify PNGase F, which was published to be purified from E.coli periplasm at 8mg/L culture

As we don't have real good experience with periplasm purifications, I am wondering if anyone among you has alternative expertise?

Thank you very much! Best

Sabine

Dear Sabine,

how about expressing a GST-fusion using a pGEX-construct as described here:

http://www.ncbi.nlm.nih.gov/pubmed/8976570

I haven't tried myself but sounds promising. Let me know if it works, I might ask you for sending the construct to us. :-)

Good luck!

Hüseyin

Hi Sabinne,

Here is a protocol I used some years ago:

1. Resuspend the cell pellet of last step (of 100ml culture) thoroughly in 10 ml of 30 mM Tris-HCl pH 8, 20% sucrose. Then add 60 µl 0.5 M EDTA, pH 8 (final concentration of 1 mM). Add a magnetic stirring bar and stir slowly at room temperature for 10 min.

2. Collect the cells by centrifugation at 10,000 × g at 4°C for 10 min. Remove all of the supernatant and discard.

3. Thoroughly resuspend the pellet in 10 ml of ice-cold 5 mM MgSO4 and stir the cell suspension slowly for 10 min on ice. During this step, the periplasmic proteins are released into the buffer.

4. Centrifuge at 4°C for 10 min at 10,000 × g to pellet the shocked cells. Transfer the supernatant (periplasmic fraction) to another tube. Avoid removing any loose pellet with the supernatant.

5. Save the cell pellet on ice for further processing of the soluble and insoluble cytoplasmic fractions in the following sections.

Use the pellet of the last step to check the presence of the protein in the soluble and insoluble cytoplasmic fraction: Resuspend last pellet in 10ml Lysis buffer + Lisozyme; sonicate; spin 15min 4C  12000rpm ; separate soluble cytoplamic supernatant from insoluble pellet.

Resuspend last pellet in 10ml buffer and take a sample for PAGE-SDS: insoluble cytoplamic fraction.

Run PAGE-SDS of: a) concentrated X10 supernatant medium  b) periplasmic fraction  c) soluble cytoplamic supernatant  and  d) insoluble cytoplamic fraction

Nevertheless, if you have a good tag, perhaps you can avoid all this, and just try your protein as a citoplasmatic protein

Mario

s 

Dear Hüseyin and Mario,

thanks for the reply! We are going to try both GST and MBP. In case it will work, you will get the clone!

Best

Sabine

Hi Sabine,

We habe obtained the GST-PNGase and GST-Endo F1 clones form Yoav Peleg (Weizmann Institute) and we were able to succesfully express these proteins using the protocol described in the paper that Hüseyin mentioned. One of them expresses somewhat better than the other, but both gave a couple of mg of protein per liter E. coli culture and both of them are active in in vitro deglycosylation assays.

Best,

Patrick

I am following this from the sideline, as we are also interested in this. I will sadly not be comming to Porto next week, but we will be represented by Stefan Kol, who is responsible for the protein purification in our project. I sincerely hope that you will all have a good time together, end I will think of you. Hopefully I can be present at the next meeting. Best, Bjørn