OpenTaq Expression from freegenes

Hi community! We were unable to obtain a significant expression of OpenTaq. We transformed BL21 cells with the plasmid sent by freegenes and induced with IPTG 0.5mM.
Have anyone expressed OpenTaq from this plasmid yet?
Javier

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Hey Javier,
Do you have pictures of your SDS gel? At what conditions did you express the protein (temperature, time, medium)?
All the best,
Kathrin

Hi Kathrin, The expression was at 18°C 16h on LB. We try 3 times, the first one, we continue the purification protocol until the end. No protein activity was achieved.
The gel show marker, Not induced 1; Induced 1 and the same for colony 2.

Thanks for the help
Javier

How did you try to purify your enzyme? Did you follow this protocol? https://drive.google.com/drive/folders/1dWxrNvJIQVnS582bt0Iu6Yvu1q5XLQXs
If not, please let me know which one you’ve used :slight_smile:
Did you alter anything?

I seems like you could have some expression (and you’ve had some leaky expression before), but to be sure you can check by Western Blot. Do you have the possibility to do that? (You run your gel in too much voltage btw, that’s why it’s melting a little. It’s not disturbing since your protein runs about 90-95 kDa).

Unfortunately, it seems like you don’t have a tag as anti-body target for staining. But there are also taq-specific ones you can order, if you have the resources.
Did you use this sequence? https://benchling.com/openbioeconomy/f/lib_LngJ7WMH-dna-polymerase-sequences-to-freegenes/seq_Dn5K8m70-taq-optimized-and-domesticated/edit

Probably we can have another meetup-call with some trouble shooting. I will try to get in touch with an enzyme purification group at my university & will suggest a new meeting.
There are also other groups, who are struggling :slight_smile:

But let’s see, if we can find some solution in the forum aswell :slight_smile: