Redissolving lyophilised proteins?
(by shifalich from rediffmail.com)
Sun Aug 23 03:01:59 EST 2009
I am expressing my protein in Origami Strain transformed with pET 32a containing my protein of interest. The protein is expressed as a fusion protein with Thioredoxin tag and His tag with enterokinase (Ek)cleavage site.
After purification on Ni-NTA column, I purify the recombinant protein on Reverse phase HPLC and eluted protein is lyophilised. Then, I try to dissolve the protein in Ek digestion buffer but the powder does not go into solution completely even dissolving the protein in appropriate buffer and pH. I loose almost 80-90% ptotrin there.
Even soncation doesn't help protein dissolution.
Can anybody suggest how do I dissolve the protein powder completely?
Second problem is:
After digestion, protein is again purified on C18 column (It is 10kDa protein). Protein gets eluted in multiple pks. My protein has 10 Cys residues and is expected to form 5 disulphide bonds.
Are these multiple pks. because of isoforms formed because of disuphide pairing?
Due to this the yields drops down to few micrograms/l of bacterial culture.
How do I overcome this problem?
I am already using Origami strain. Do I again have to denature, refold and purify thr protein again?
Does anybody have a protocol that they have tried and works for such small proteins with so many disuphide bonds? Please share with me.
Please send your responses even if you can help in soving even a single problem out of all above.
Your kind help will be highly appreciated.
On Tue, 04 Aug 2009 23:49:13 +0530 wrote
You might consider immobilizing anti.myc antibodies on an activaed
sepharose (eg CNBr, NHS). Should not cross-react with endogenous myc.
On 4 Aug., 15:08, Chris McDermott-Roe
> Hi all,
> I have produced a myc-conjugated fusion protein and hope to express it in
> mammalian cells and subsequently purify it. Are there any myc resins out
> there that would permit large-scale purification of my protein? Second, if
> I was to transfect my protein into say HEK293 cells, would I need to worry
> about co-purifying endogenous m-Myc if using such a resin?
> Thanks all
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