I am expressing Leishmania protein in bacterial system. This sequence has four rare codons of proline, isoleucine, leucine and arginine. I have tried BL-21 DE3, BL-21 Gold, BL-21 RIL, BL-21 RP, Rosetta and P-lys cells. But in none of the cells I am getting induction. I have checked sequence of complete pET 28b cloned required DNA fragment .I have tried different IPTG concentration (0.5mM,1mM) and induction temperature (37 and 25 degree celcius overnight). I have also changed IPTG. But still waiting for induction to be occur. Please take me out from this difficulty.
If your fund supports, you can use codon optimized synthetic plasmid.
all of the proteins can not be expressed successfully in E. coli. first you have to check the expression level at 37 degree using more than 1mM of IPTG conc. (2mM and mM). if you are not getting success change your vector like pet30b, pet 32a and pet 42a instead of pet 28a.
Are those four codons rare in E.coli or Leishmania ?
The Frydman Lab has published some interesting observations concerning rare codons and proper protein folding. You might want to take that into account.
doi: 10.1038/nsmb.2919
Hi Stefan,
These four codons are rare in Ecoli BL-21 strains what I have mentioned above. Now I am trying BL-21 RIPL cells for protein expression. Lets see what will I end up with.
Hey, i have expressed a human protein (with a lot of rare codons) in E. coli. According to my many years experience, rare codons did not impair the expression of that protein, instead i encountered many truncation products. Also, please mind that rare codons pose problems at the translation level. One can try northern blotting or other mRNA detecting strategies to ensure the transcription of the protein. According to my gut feeling, something is wrong with yours vector. Please check the IPTG stock (put a control) as well as antibiotic stock. You need to add appropriate amount of antibiotic to select for the positives. Also check the growth curve of transformed and non-transformed bacteria (may give a clue, if protein is toxic to bacteria). By the way, what is the length of the protein you wanna express? best wishes for the design of further experiments.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression