I have cloned a rice gene into the pET28a His tag vector. I have confirmed recombinance by sequencing and it is in frame. I run the SDS PAGE along with my empty pET.
I am attaching my gel here.
Lane 1: Protein marker, 2: pellet from uninduced empty pet 3: supernatant from uninduced empty pet 4. Pellet from induced empty pet 5. Supernatant from induced empty pet 6. Pellet from uninduced pet with insert 7. Supernatant from uninduced pet with insert8. Pellet from induced pet with insert 9 supernatant from pet with insert (my protein size should be around 20kDa)
Hi Shanice,
After a quick search online I think you might be using the XXL Deluxe marker from Geneon. If that's correct, then the bottom band (green) is 10 kDa, the next one up is 17 KDa and the next one up is 26 kDa. Your protein should be visible in between the second and the third marker bands from the bottom and it look like it either hasn't expressed at all or there is so little of it can't be seen through the native E. coli proteins.
Gabriela is right and you should check if you protein is being expressed with a western blot. Since your protein is His-tagged, that should be easy enough, just use a primary anti-His-tag antibody, which will give a very strong signal even if the protein can't be seen on the gel with the naked eye.
What was your expression protocol? (cells you used, temperatures, IPTG concentration, etc...)
It could also be a problem with the tag. Sometimes N-terminal His-tags interfere with the correct folding of a protein for various reasons. This can generally be rectified by trying a C-terminal His-tag instead. At this stage I wouldn't try a solubilisation tag (SUMO, MBP, etc...), as these are used to make insoluble protein soluble and it doesn't look like you have insoluble protein either.
If playing around with the expression protocol and the tags still doesn't work and your sequencing is definitely correct, then check for rare codons (I don't think rice should have any ... but I haven't worked with plant genes before). If it has rare codons, then choose an expression strain that codes for these codons (e.g. Rosetta 2 codes for most rare codons) or, if that still doesn't work and you're sure it is the codons that are the problem, then have the gene synthetically codon-optimised, which will produce a gene that uses the normal E. coli codons instead of the native codons from your rice gene.
I hope that helped,
Tony
Dear Shanice,
Better to attach SDS-PAGE gel pic so that we could understand easily your experiment bcoz there are so many reasons for that.
Best wishes
Radhey
Hi Shanice, I would like to know wich band of your protein ladder you are considering as 20kDa. For the majority of protein ladders it should be the third or fourth band (down to up), if this is the case you are not having any production of your protein, or perhaps a little bit but you will have to confirm by western blot. Did you check for rare codons? maybe that is the problem. I do not know how did you prepare your gel but if you use a 4-20% gradient gel you will have a better resolution.
Best!
Gaby
Dear Shanice
Thanks for pic. but there is some lack of information in the gel like your protein ladder is not marked so I unable to figure out the expressed protein band.
There are some points which I mention here to clear out from you....
1. What is the actual size of your concerned protein?
2. How many times you have repeat this experiment?
Best!!!
Radhey
Hi Shanice,
After a quick search online I think you might be using the XXL Deluxe marker from Geneon. If that's correct, then the bottom band (green) is 10 kDa, the next one up is 17 KDa and the next one up is 26 kDa. Your protein should be visible in between the second and the third marker bands from the bottom and it look like it either hasn't expressed at all or there is so little of it can't be seen through the native E. coli proteins.
Gabriela is right and you should check if you protein is being expressed with a western blot. Since your protein is His-tagged, that should be easy enough, just use a primary anti-His-tag antibody, which will give a very strong signal even if the protein can't be seen on the gel with the naked eye.
What was your expression protocol? (cells you used, temperatures, IPTG concentration, etc...)
It could also be a problem with the tag. Sometimes N-terminal His-tags interfere with the correct folding of a protein for various reasons. This can generally be rectified by trying a C-terminal His-tag instead. At this stage I wouldn't try a solubilisation tag (SUMO, MBP, etc...), as these are used to make insoluble protein soluble and it doesn't look like you have insoluble protein either.
If playing around with the expression protocol and the tags still doesn't work and your sequencing is definitely correct, then check for rare codons (I don't think rice should have any ... but I haven't worked with plant genes before). If it has rare codons, then choose an expression strain that codes for these codons (e.g. Rosetta 2 codes for most rare codons) or, if that still doesn't work and you're sure it is the codons that are the problem, then have the gene synthetically codon-optimised, which will produce a gene that uses the normal E. coli codons instead of the native codons from your rice gene.
I hope that helped,
Tony
Tony's answer is very complete. Remember E. coli does express many endogenous proteins and some of these will be around 20 kDa. Due to the low levels of expression relative to endogenous E. coli proteins you will need to do a Western to see if your desired protein is over expressed.
Hi all,
Sorry for incomplete information. My expected protein size is 20 kDa. The bottom band (green) is 10 kDa, the next one up is 15 KDa and the next one up is 25 kDa. I am using BL-21(DE3) and Rosetta (DE3) for expression. My expression protocol is as follow: 25C (4hr), IPTG concentration 0.3mM. What is the best way that I can check for the rare codons?
Thanks for all the useful suggestions. I will try to conduct a western blot.
Dear Shanice
Thanks for information. According to my observation your protein is not expressing as seen in the gel with respect to ladder.
There are some suggestion for you...
1. Check your sequencing result carefully.
2. Translate your sequencing result into amino acid form through Translate tool (Expasy server) from vector initiation start site (Met, NcoI) to check your concerned protein is in frame.
3. Check rare codons in translated protein sequence (http://nihserver.mbi.ucla.edu/RACC/)
4. Increase IPTG conc. upto 2mM and temperature 37degree.
Try and clear out the above exercise. If u want any help then let me know.
Best
Radhey
Hi Dr. Radhey
Sorry as I misinterpret the results earlier. Thanks for your useful feedbacks. I have done the first and second step prior the expression. I am quite confident that they are working.
I am using Rosetta (DE3) and all the rare codons are well covered. I will try to increase the IPTG and the temperature as you suggested. Alternatively, I will also work on the Western blot.
Hi do a dose response of induction with various concentraions of IPTG; Check if the induction changes. This looks like some host cell protein. Just check this in other expression host. Good luck !!
Dear Shanice
If u tried in all way then simply change the vector as pET28a vector is low expression vector. If you feel low expression of your concerned protein then simply do the western blot or directly apply the supernatant of inducted sample on Ni-NTA His tag coloum for final conclusion but I don't think so it will works.
You use pET15b/16b vector as these vectors are high copy plasmid vectors.
Best
Radhey
This protocol generally explain expression recombinant protein in Ecoli.
https://www.neb.com/tools-and-resources/feature-articles/bypassing-common-obstacles-in-protein-expression
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