No protein expression after IPTG induction - No protein expression after IPTG induction (Aug/15/2005 )
Hi,
I wondered if anyone else has had this problem and/or had any solutions:
I have cloned a 28 kDA gene into a Novagen pet21 vector. After induction with IPTG there is no detectable protein expression - nothing on gels in supernatant, cell pellet nor on gels after attempted Ni-bead enrichment. There is no expression even when induced at different phases of growth and even in Rosetta2 cells with the extra rare codons. The protein is an E. coli protein.
This is a stupid question, but have you sequenced the insert? Is there any chance a stop codon was introduced during cloning?
I have had similar problems in the past. When I had the problem, it was with a cerevisiae gene being expressed in Rosetta. I fixed the problem by examining the codon usage - although Rosetta catches the most rare codons, there are still some mid-level codons it doesn't complement. A single one of these won't prevent expression, but two or three in a row will stall the ribosome just enough to prevent expression of the full-length construct. That could be the problem, but I'd have a hard time believing it since it's from coli in the first place. If you want to be sure, check codon usage with the following site...
http://www.gcua.de/
If you see runs of three or more in the <15% usage range, you could try switching them out via mutagenesis.
Another suggestion is to try expression in pLysS. Sometimes it helps when attempting expression of a toxic species - the construct may turn lethal when there's too much of it, and the bacteria may adapt by getting rid of it as soon as it's expressed. Also doubtful, but might be worth a shot.
Hi, thanks for replying.
The insert is sequenced and is correct. The gene is packed full of rare codons which is why I tried using the Rosetta cells. Have you found that even in Rosetta cells the rare codons continue to be a problem?
T7 promoter is very tricky!!
Media is important sometimes.
For example, my protein was expressed in M9. but not in LB.
Did you check the growth phase after OD600=1.5?
Another protein was expressed only when IPTG induction happened at OD600=1.3 or later.
Also you should consider the lethality of the expressed protein, as mentioned above.
However, I recommend to use another expression vector other than pET series. I had a lot of problems with expression and solubilization with the vectors.
Good luck anyway.
I am going to try M9 media as you suggested.
I did try to induce after the cells reached stationary phase - still no protein expression.
I also tried pLysS strains which didn't help.
What other vector series would you suggest?
The insert is sequenced and is correct. The gene is packed full of rare codons which is why I tried using the Rosetta cells. Have you found that even in Rosetta cells the rare codons continue to be a problem?
They can be. Bear in mind that standard Rosetta only complements 6 rare codons (AUA, AGG, AGA, CUA, CCC, GGA). Rosetta 2 includes two more, I think. If you run your sequence through the gcua site, you should get a chart readout showing the codons. If you find runs of low-usage codons (again, in the <15% range, 3 or more in a row) that aren't complemented by Rosetta, that can cause a problem in expression, and you may be able to fix the problem by engineering silent mutation to the more common codons.
It typically will only matter in the early part of the sequence (say, the first 40-50 codons). The ribosome is a little more finicky with short constructs, and is more likely to fall off during the early stages of transcription. Thus, later in the transcript, when you've synthesized a large part of it, transcription is less likely to terminate, so you shouldn't need to be exhaustive with silent mutations.
Another option you can explore is Origami cells (combined with Rosetta to make Rosettagami). Origami has mutations in thioredoxin reductase and glutathion reductase, so disulfide bond formation is enhanced in the cell. If the fold of your protein is critically dependent on disulfides, this strain may help it fold to a stable state and prevent degradation. That's assuming degradation is occurring, of course.
I did try to induce after the cells reached stationary phase - still no protein expression.
I also tried pLysS strains which didn't help.
What other vector series would you suggest?
So Anoush, were you able to express your protein ? If so please let me know the catch. I am also having a problem in expressing a protein design(containing 2 rare arginine rare codons) in E. Coli, using pET19b expression vector in Rosetta(DE3). I tried 2XYT and LB media and normal expression protocol for 4 hours at 37 deg. SDS shows light 2-3 bands, which looks similar with another design having 2 repeats of the sequence. Any expert comments is appreciated.