I am trying to express fusion peptides of mycobacterium tuberculosis in BL21DE3 and Rosseta DE3 but not getting any expression. Vector I have used is pET23a and pET28b. Gene is fusion of 6 different peptides. I have used different temperature, time and different concentrations of IPTG, still not getting any expression. there is no secondary RNA structure. What could be the problem behind that.
Hey Sudeep!
Do you already evaluated the expression using western blot against His-tag? or using Northern blot?
In many cases peptides are very toxic to E. coli. Maybe you can try to use other E. coli strains, like C43, specific to toxic proteins, and perform the overexpression experiments at very low temperatures with short inductions time (2h).
Good luck!
When you write that there's no secondary RNA structure then you mean that there's no or minimal GC content in the 5' end of the DNA gene sequence? Do Mycobacteria use preferred codons different than E. coli? What are the cell morphologies post induction? Are the peptides fused to a cleavable protein and does it matter whether it is the N or C terminus? Usually peptides don't solubly express well or purify well from E coli.
I'd agree that a western blot would be an excellent diagnostic in this case, assuming that you have some fusion to test against. M. tuberculosis has a very biased codon usage (very high GC), so if you have not codon optimised for E. coli, that might be an issue. Rosetta will help, but may still not be able to cope.
Good luck!
I have tried western blot with His tag but did not get any expression. Yes the gene is from mycobacterium but the gene is optimized for both E.coli as well as mycobacterium. Both are not getting expressed.
How long time your cells have been transformed with the plasmid?
I worked with some proteins that whenever I needed to do an expression experiment I had to transform the cells again because my glycerol stocks did not work.
Do you ever tried it?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides