No activity of fungi protein in E.coli - (Oct/13/2011 )
Without some control to verify expression, you can't be sure that you are actually expressing enough protein to see activity. If you cant be sure that the band you see is your protein, and you have no activity by enzyme assay, then you either need to have an empty vector protein extract control or do a western blot for TSase. I know that there are commercial antibodies available for TSase, but not sure if they are for the same species as you are working with.
If the other group that you were working with was successful using the pET vector system, and you are having trouble with a constituative promoter, it is possible that your protein expression levels are not high enough or that your protein concentration in the crude extract is not high enough to give detectable activity. The pET system produces very, very high levels of protein in a short time, in a smaller number of cells. You could just be having a concentration issue, especially if you cant directly observe your band in an SDS-PAGE. Most proteins expressed in the pET system will show up as a huge blob on SDS-PAGE of crude extract.
Lastly, did the other group show activity in crude extracts? If not, it is possible that there are inhibitors present in the crude extract that were removed during the fractionation performed by the other group.
Best of Luck.
allynspear on Thu Oct 20 12:15:28 2011 said:
Without some control to verify expression, you can't be sure that you are actually expressing enough protein to see activity. If you cant be sure that the band you see is your protein, and you have no activity by enzyme assay, then you either need to have an empty vector protein extract control or do a western blot for TSase. I know that there are commercial antibodies available for TSase, but not sure if they are for the same species as you are working with.
If the other group that you were working with was successful using the pET vector system, and you are having trouble with a constituative promoter, it is possible that your protein expression levels are not high enough or that your protein concentration in the crude extract is not high enough to give detectable activity. The pET system produces very, very high levels of protein in a short time, in a smaller number of cells. You could just be having a concentration issue, especially if you cant directly observe your band in an SDS-PAGE. Most proteins expressed in the pET system will show up as a huge blob on SDS-PAGE of crude extract.
Lastly, did the other group show activity in crude extracts? If not, it is possible that there are inhibitors present in the crude extract that were removed during the fractionation performed by the other group.
Best of Luck.
Hello allynspear,
I am still focusing on this problem. Recently, I tried to express this protein with a strongest constitutive promoter we have, and cultivate them under different temp (25, 30, 37 degree), but there is no activity at all. We tried western blot to check His-tag protein in pellet and supernatant, but no band is present. It is really strange. There is no reason that it can not express any proteins. I will re-do western blot to confirm it.
Additionally, I transfer my gene into pTrc vector (not pET vector). I suspect that protein expression is so dependent on the promoter, even I just want to see a little activity first.
Biogareth
Dear Biogareth,
can you give us the link to the Japanse paper you are referring to? ...i would like to know what pET vector they are using?!?!?!
Maybe your protein contains disulfide bonds and does not fold correctly in the cytoplasm and was therefore secreted to the periplasm by the Japanese people to allow for proper folding?
Regards,
p
pDNA on Sat Nov 19 15:58:55 2011 said:
Dear Biogareth,
can you give us the link to the Japanse paper you are referring to? ...i would like to know what pET vector they are using?!?!?!
Maybe your protein contains disulfide bonds and does not fold correctly in the cytoplasm and was therefore secreted to the periplasm by the Japanese people to allow for proper folding?
Regards,
p
The link:
http://www.ncbi.nlm.nih.gov/pubmed/9763690
pET16b has no leader for secretion ...so we can rule that problem out!
Have you ever checked plasmid stability? ...maybe, since you use a constitutive promoter, there is strong selection pressure towards plasmid free cells or mutations that abolish production of your gene of interest. I would use an inducible system (and stick to the protocol as much as possible!).
Another thing is that they assayed the activity after purification with a Ni-column ...maybe your enzyme is not active in the crude extract? ...or did i got something wrong and you also purified your enzyme?
Regards,
p
pDNA on Sat Nov 19 22:50:11 2011 said:
pET16b has no leader for secretion ...so we can rule that problem out!
Have you ever checked plasmid stability? ...maybe, since you use a constitutive promoter, there is strong selection pressure towards plasmid free cells or mutations that abolish production of your gene of interest. I would use an inducible system (and stick to the protocol as much as possible!).
Another thing is that they assayed the activity after purification with a Ni-column ...maybe your enzyme is not active in the crude extract? ...or did i got something wrong and you also purified your enzyme?
Regards,
p
For expression of this enzyme, Janpanese guy found that the similar amount of protein was expressed with or without IPTG by pET16b vector. It is quite strange, and I don't how to explain this phenomenon. Does it mean that the strong promoter pTrc dosen't influence the expression of protein?
In addition, if purified enzyme has the activity, I think crude enzyme should have activity as well. No inhibition compound was reported.