Hi Firdous,, As you mentioned they are many things to change and try. before changing vector it is more efficient in my opinion to change expression conditions (temperature, amount of IPTG, length of culture), culture medium (regular ones against M9 or auto induction media) and expression strains BL21, BL21gold C43 and and BL21 Lemo cells.
Then you can change vector (arabinose or rhamnose controlled promoters) and fusion (MBP or GST maybe). Leaky expression can have deleterious effects on expression levels and culture stability/degenerescence (especially dependent on the type of antibiotic you are using). Tightening up the expression control maybe important in the case of a toxic target). BL21 Lemo cells can be very useful in those cases. If your gene has an odd codon usage (compared to your expression host) you may want to think about recoding it or use rare tRNA plasmids to try to compensate for that. The fact that you mentioned that you see the Sumo-his but not the following target protein is worrisome. Since you mentioned the vector is fine I would think that there is a problem of translation/folding of the target sequence, maybe check the presence of rare codons that could promote pausing for example. I don t know how easy it is to check for sequences that would affect the local structure of the mRNA but codon optimization programs should take this into account. Good luck On Sat, Dec 9, 2017 at 5:27 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 > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu
