Hey Anamika I know SH2 domain is very much well-studied domain, and i am not sure why are you facing troubles in the expression of this protein. Just go through the literature and read about different protocol of expression of SH2 domain from several different proteins.. Well, reading that you have "inconsistent" expression suggested me something else. Do verify the IPTG stock. Also, confirm the expression via western using anti-His antibody. You may also check the prevalence of rare codon in the proteins. If present, then Rosetta would be a better option than DE3 cells. Eventually, the best would be to follow the established protocol from the closest-homology SH2 domain, and move from there onward.
Best wishes -Z Zaigham M Khan, PhD Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York On Thu, Jul 6, 2017 at 10:11 AM, Anamika Singh <[email protected]> wrote: > 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 >
