Hi friends
I have one protein that have difficult process(use 2me, change pH up to 11.6 and some changes in induction step) to solublizing it in supernatant. after change pH to 11.6 my protein binding to column(Ni-NTA) decreased and half of my protein washed in washing buffer.
(Protein PI is 8.6)
what can i do?!
any solution?
thanks
Derr @Alireza
2 questions:
1) Why are you using soo high ph as 11.6?
i tihbnk that with pI 8.6 a pH 7 or 10 could be ok! 11.6 is little extreme and i'm not sure it will not affect the protein conformation and the histidine binding to the Nckel.
2) why are you using 2me? Because it is contains many cysteines and it is insoluble with-out it?
Did you tried to run the protein in the elution and in the washing in SDS-page with out reducing agent? Because is it possible that you have protein aggregates where the His-tag is not well exposed.
best regards
Manuele
hi Manuele Martinelli
thanks for your answer
my protein was totally insolouble
after change induction condition like time(20h), temp(15c) and using low iptg
and using 2% 2me and high pH in Lysis solution(8M Urea, NaCl, Na2HPO4)
the soloubility of my protein increased up to 40%
in lower ph like 10 and 7 my solubility is low about 20%
i think your last question maybe happen. because after collect washing (my protein + non specifics) and repurify it my protein came out in flow-throw and washing. but i am using 8M urea...
Try to load the fractions in SDS page with out reducing agents and see If you have high mw aggregates.
How many cysteines are present in your protein?
Does your protein contain any hydrophobic regions (eg transmembrane region)?
best regards
Manuele
Manuele Martinelli
thanks ok i test it.
my protein is single chain anti-CD22 that have 246aa with 4 cys residues
Dear Alizera
If you are working with antibodies (single chain or fab) i'm quite sure that the test will show you the formation of high molecular weight aggregates with out reducing agent.
If you would like to produce a good single chain, you need its S-S bond are correctly formed, because it is essential for its function and therefore you have to find a condition that allow you to produce it in soluble way with out any 2ME or other reducing agent.
To produce folded and functional single chain, i suggest to you to add a pelB or OmpA leader peptide to your sequence and express it in the periplasm where the red-ox potential is oxidizing and the formation of S-S bonds is permitted.
in the past i purified hundreds of single chain antibodies in this way and it works. The expression yields where not amazing but using an high cell density media (as the Enpresso and YGR we were able to recover small amounts of Scfv required to perform ELISA and Gyrolab analysis from 8ml culture)
Attached you can see a brief summary of it.
We used pet vector supplied with an Ompa leader peptide and standard BL21(DE3) strains. We performed some test also with the BL21(DE3) delta TolR strain to produce and recover the single chain from the OMV released into the media. The primary results obtained in small scale were encouraging but we did not had enough time and resources to develop a good protocol.
You can try to use also the BL21 Origami or the T7 shuffle strains (with a less reducing cytoplasm) with your current clone and see if you can obtain some soluble single chain with-put 2ME but i was not able to obtain good results with it.
if you are interested you can find more information about YGR and Enpresso at the following link of my blog (ProteoCool);
https://www.blogger.com/video.g?token=AD6v5dxhZKT6Tvug0rLIZgcg8nyl5CwzQSFiX9pLglN-foSxrhnBjoEr7ReFTWj5bbeiloMCNhNoJn2HgHEgR2eC7SYwl5TPVwlCYNniPhSpeELHsevHk1dREBGIdHqskofd9HgX-2gr
and about non reducing SDS-page at the 7'30'' of the following link
and about non reducing SDS-page at the 7'30'' of the following licx4RQ0mfxIRjK1x1YHBCYw3Av9JZTXdeO6JXLu6wl6Cd2omOIASqXY7BDpDcvlzrJ8fQSoqXnMyd5Fqcc3vRe-1JCCyISDv7Y-UACmOPKhEV6M
Good luck
Manuele
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