Hey Firdous Yes, too much information can be confusing. I have expressed and purified over dozen of different proteins. I usually found in literature the protocol for the expression and purification of another homologous protein. That is the best way to start in my opinion. Some folks may want to invent the wheel one more time, and i have no problem with that either....
I got more excited with your first line that you can see the tag, and no protein. It seems as (A) you got an empty vector in the bacteria; or (B) the fusion construct is self-cleaving like with intein. you may wanna check the vector itself. Best wishes for the experiments! -Z Zaigham M Khan, PhD Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York On Sat, Dec 9, 2017 at 8:27 PM, Firdous Tarique <kahkashantari...@gmail.com> wrote: > Hello everyone. > > I am struggling with the solubility or expression of my proteins in > BL21DE3 E.coli expression cells. In one of my construct I have a Sumo-His > fusion at the n-terminal of my protein. After induction what I see is the > expression of the Sumo tag only. My sequencing results are fine and there > is no mistake in cloning. I wonder why I am seeing only the expression of > the Sumo tag although my fusion protein is in frame with this tag. > My second question is related with the very low expression of one of my > gene. A brief literature search suggests so many things to improve the > expression like changing the host, vector, media etc. It is a nuclease and > cloned in vector with a Sumo-His tag on it. I am able to purify the little > amount from E coli BL21DE3 host. The problem is related with low expression > which is clearly observed in difference in the pre and post induction > lysate on SDS PAGE. Out of so many option available in the literature I am > confused what to try first. Any general idea? > > Your suggestions can help a lot. > > Kahkashan > Ph.D student > Delhi University > India >
