OpenVent purification

Open DNA Collections Enzyme Expression Protocols
1

Hi all.

We are producing OpenVent-mCherry at my lab and we would like to try an AKTA purification instead of the classic Bench purification. Does someone have tried this? In that case, would you be able to share the protocol?

Thanks!

Pablo

2

Hi Pablo!

We don’t have OpenVent-mCherry but we purify diferent chromoproteins (fuGFP, aeBlue, Yukon, Dasher…) by AIEX in an AKTA Go. We follow this procedure. We adapted the flow rate and CV to our system. If you want I can share you a gradient and a step elution chromatogram so you know what to expect

3

Hi Jon.
We attempt to purify the protein by IMAC, however, I´ll take a look to the procedure you´ve provided. Thank you very much!

Pablo

4

For IMAC purification we have found that simple Ni-NTA columns are not enough to get pure protein and we think a second step using heparin columns could be useful but we haven’t tried it yet.

FYI: Bst.LF on the left after Ni-NTA purification (for comparison). OpenVent-mCherry on the right after one step purification on Ni-NTA. Yield is super high though, 5-10 mg/mL on Bradford.

IMG_6139.HEIC
IMG_6139.HEIC2141×1275 134 KB

Also looks very…mCherry :stuck_out_tongue:

IMG_6133.HEIC
IMG_6133.HEIC3024×4032 1.8 MB

IMG_4325.HEIC
IMG_4325.HEIC3024×4032 2.5 MB

IMG_4332.HEIC
IMG_4332.HEIC3024×4032 2.31 MB

Let us know how it goes for you!!

1 Like
5

Thank you so much Sev!
We´re just about to try the NiNTA purifiaction. I´ll let you know how it goes.
Did you use any desalting or buffer exchange step after purufication? In that case, which final storage buffer did you use.

Cheers,

Pablo