How to separate a chaperone from my desired protein?

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Jerry999
Jerry999's picture
How to separate a chaperone from my desired protein?

Hello,

I have a problem to purify my protein. It is expressed by bacurovirus and Hi5 cells. This protein secretes only when chaperone protein Calreticulin is co-expressed. My problem is that calreticulin tightly binds with my protein after anti-Flag column and gel filtration. How can I separate them? Does a detergent work? Thanks a lot.

Jerry

qiang wang
qiang wang's picture
try first add Urea grains to

try first add Urea grains to your solution to a final concentration of 8M, which should be able to separate the two proteins, then do the purification, and dialysis the urea out afterwards

wrote:

Jerry999
Jerry999's picture
Thank you, qinglongyanyuedao.

Thank you, qinglongyanyuedao. I am worrying about if 8M Urea will make my protein unfolded or misfolded. I will try to use a lower concentration of Urea.

Jerry

R Bishop
R Bishop's picture
Jerry,

Jerry,

Have tried adding a chelator like EDTA or EGTA? Im pretty sure these will break any interaction with calreticulin and polypeptides.

check out this paper
Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD

In the methods section they use EDTA to elute calreticulin from a peptide column.

"The elution of the impure protein was performed with 3 mm EDTA in 0·6 m NaCl/Tris-HCl buffer"

Rusty

Clin Exp Immunol. 2003 October; 134(1): 143–150.
doi: 10.1046/j.1365-2249.2003.02246.x.

Jerry999
Jerry999's picture
Thank you, Bishop, I will try

Thank you, Bishop, I will try to add EDTA and then run a native gel to see if the two proteins can be separated.

Jerry

qiang wang
qiang wang's picture
Jerry999 wrote:Thank you,

Jerry999 wrote:

Thank you, qinglongyanyuedao. I am worrying about if 8M Urea will make my protein unfolded or misfolded. I will try to use a lower concentration of Urea.

Jerry

yes, 8M urea will unfold your protein, but it will be refolded after dialysis out the Urea. and under most cases, 4 M Urea would be enough