I have been trying since long to express two proteins of eukaryotic origin in E.coli but somehow these proteins are not showing any expression. Even i have checked the transcript level transcripts are also not formed so can any one suggest me some protocol to at least get a decent expression of these proteins.
after checking with online tools for detecting disorderliness of the protein I have checked with tools like DisEMBL, DIsprot, PONDER. and these proteins are showing high degree of disorderedness.
Hi Steingrimur, thanks for your query to check the toxicity of proteins i have done induction in the presence of glucose also. but nothing works.
Hi Isha, I don't understand why glucose should change anything.
If overexpression of these proteins are toxic to coli, they will die. A lack of inclusion bodies in the bugs with your protein is an indicator that the cells cant overproduce it.
Have you looked into baculovirus vectors to infect sf9 or other insect cells.
Hi Isha,
Absolutely you should double-check that your protein sequences are in the right frame with the promoter in your expression vector first, if you've confirmed that you are not seeing your expected transcript levels increase after induction. I've been off by one base before, it was frustrating but easily solved.
We routinely express IDPs in our lab, one has no tag (pET-24a without the His-tag), and the other with a GST-tag with a thrombin cleavage site between the IDP and the protein. Also, there are several strategies for expression of cytotoxic proteins, including forcing into inclusion bodies/inclusion body prep, or directing the protein into the periplasm, or co-expression with a known binding partner to sequester the cytotoxic protein, for example. What is actually happening after you induce? What do the cell pellets look like afterward?
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression