I am trying to clone 2 Kbp prolyl endopeptidase gene into pET28a vector, after successful cloning into vector and transformation into BL21 (DE3) or in B834, it was checked by colony PCR and Restriction Digestion analysis, which confirm presence of my insert. The reading frame is also correct, all these are correct. My problem is when I try to induce, after induction using IPTG at 100mM conc at 0.6 OD for 20hrs, at 16C I am getting in both similar band pattern, the induced and control protein. I am not able to detect on SDS PAGE. Please suggest if any one could help me out with this problem.
May be decreasing of IPTG concentration will help. Another option is using of a set of different expression strains (Shuffle T7, SoluBL21, LemoBL21, etc).
It's not work on low concentration of IPTG, I have checked it on different concentration of IPTG
http://onlinelibrary.wiley.com/doi/10.1002/0471140864.ps0523s56/pdf - protocol of autoinducition. Hope, it will support to solve your problem.
inducing at 37C for 2-5h may guide you on expression status. if you note a difference in the expression levels then you can go for lower temp induction. good luck!
Try this.
Prepare starter culture: Single colony inoculate in Rich medium- LB or 2xYT grow 4-8hrs at 37C. Use this to inoculate the starter culture. Incubate at 30C or below for overnight until the OD600 is 0.4-1. Then store culture at 4C and use this to inoculate main culture. For T7-lac promotor IPTG – 0.2mM is recommended (or 0.05 to 2.0mM can also try) and induction temperature 30C for 5-6hrs or 25C for overnight.
Problem may be due to:
-orientation of the sequence insert in the vector – Check it!
-E.coli doesn’t do large protein all that well
- verify the gene is even transcribed in your system – do Simple RT-PCR!
- protein is present in very less amount couldn’t able to detect in SDS-PAGE – Column based His tagged Protein purification system!
Ellango, still it is not working. It gives substrate activity in Rosetta but not seen on 10% SDS gel. it also not comes in purification
What type of purification method did you use? I have similar experience recently but when I purified the protein, I could clearly see my protein band
his tag method, now I have sorted out my problem. thanks to all for your valuable suggestions.
Hello
Could you please tell us how you sorted your problem? I am also going through similar phase, not yet solved it. Jitendra Kumar
Thanks
Hi Jitendra Kumar
Kindly share how you solved your problem. I ma also going through similar problem when using pET28b plasmid and transforming it in E.Coli BL21DE3. Please share.
Thanks
Jayanti
Hi Jitendra Kumar
Please share your findings about this issue.
Thanks
Jayanti Vaishnav, were you able to troubleshoot the issue? Could you please share your inputs if you were able to sort it out?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes