Hi Anamika,
Proteins with high sequence identity can behave very differently when
expressed in bacteria. Sumo (or other tags) may or may not help. If it does,
you can express your SH2 with a tag and remove the tag after purification if
the tag is a problem for functional studies. The vectors described in this
article <https://link.springer.com/article/10.1007%2Fs12033-017-9998-6> may
work for you if you cannot figure out why you get inconsistent expression.
YZ.
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Anamika
Singh
Sent: Thursday, July 06, 2017 7:12 AM
To: [email protected]
Subject: [ccp4bb] off topic
Hi,
Is anyone has worked with STAT1 proteins?
I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was
no expression so far or rather say inconsistent expression. Sometimes the
expression was in inclusion bodies. I have tried different methods to pull out
the protein from inclusion bodies using urea, guanidium chloride tween20 but
none of them worked well. The yield was very low (very faint band on SDS-PAGE )
from 3-liter culture. I changed the host cells from BL21 to Rosetta DE3 cells
but no success so far.
We thought to use some other vector system like with SUMO tag but did not
proceed because the aim of the project to design inhibitor and tag will
interfere.
Please suggest me something so that I can complete my project in lesser time.
Looking forward to valuable suggestions.
Thanks
Anamika
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