I was curious if anyone of your labs have experience with any of the SUMO proteases that are commercially available? (Also, at which ratio target to SUMO they can be used).

Alternatively, I am interested in producing SUMO protease in our lab, if any one are willing to share a MTA protected plasmid.
AT this point, however, we only need a test batch, so we could go with the commercial.

-Gro
Norstruct /Tromsø

Dear Gro,

I received the vector from Husseyn, and it cuts very good at 1:1000 at 4C ON

Of course that 1:1000 depends of the construct, the concentration, etc Nevertheless it is a fantastic yield. It is a very easy to prepare and remain active after a long time at -80C

Mario

Hi Gro,

The prices from commercial sources have not dropped so much but there are also different versions availble now with different specificities for the different SUMO tags available, 'star' and 2 versions that may be more appropriate for you. I have not used the protease from commercial sources because we have the clone for expression.

We amplified it from S.cerevisiae genomic template in Oxford and it was then cloned into a pTriex background with an N-terminal his tag. The clone expresses very well in E.coli (I can check the yield of our last prep for you) , IMAC/SEC gives sufficient purity and we can normally use it here in BCN at 1:50, 1:100 protease: protein ratio (it is so easy to make we are a bit reckless with the ratios!). We have not used it as much as e.g. 3C but my impression is that it is possibly less 'context' sensitive than other proteases.

Contact Ray in Oxford for the clone as I cannot distribute it.

Hope that helps

Nick

I'm overwhelmed by the rapid reponses, Mario and Nick - thanks a lot. Since you're all agreeing that it sooo easy to make (wonderful!), I will just go directly for a lab made version, also considering that the commercial proteases still seem very costly. I'm already in contact with Hüseyin/EMBL.

Sounds good using 1:1000 as a starting point, to further test empirically, Mario.

-Gro

Dear Gro,

we obtained SenP2 from Christopher Lima (MTA signed).

As our colleagues mentioned it is extremtly active and also robust in the presence of Urea etc (Hüsyein gave a talk in Barcelona)

Best

Sabine