I am trying to express a 48 kDa protein in a pEt28a expresison vector. Attached is the map of the construct. I have tried expressing my protein in small aliquots just to see if my expression works but I see no overexpressed protein band in my SDS gel. Apart from that, I've checked my construct with PCR and definitely see bands so, I know my construct works. I have tried inducing the protein with 1 mM of IPTG at 37C for 3 hours but that didnt work. I am thinking to try inducing at 16C overnight with 1 mM IPTG, and 25 C overnight with 1 mM IPTG. Am I missing out on anything?
Liyana, you planned it correct. You may check expression at 16 or 24 degrees with over night incubation. If even this does not work try using different medium like TB, AIM if the expression host is E.coli. In some cases you wont see huge over-expressed bands, even if you see a thin band you may try purifying the protein using 100ml culture.Good luck!
Hello Liyana,
You can try doing small-scale expression test in 24 well plates using four different strains in four different media (LB, SB, TB, M9) to check which strain would be the best and the media. Once you know that you can use same strain and media and do comparision between IPTG- Induction and Autoinduction.
I agree with the comments given above. In addition, I need to raise some questions for you to thing about them. Do you have any idea about the physico-chemical properties of your target protein, such as hydrophobicity? If your protein is more hydrophobic, the cell will devise a mechanism for premature termination or degradation of your proteins in order to prevent accumulation of target proteins in cells. Bye the way, how is your mass cell? Did the cell grow well under the conditions you used for your protein expression? Did you use fusion tags? I recommend the use of appropriate tag proteins for enhanced expression of soluble proteins.
If my target protein is hydrophobic, and it is just expressed very small amount after induce, even no difference comparing to not induce. I check it by Western blot also. I tried several condition as changing the amount of inducing agent or temperature and time, but they did not work. And I used BL21-AI and also tried with BL21-DE3. What should I do?
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