from pet-SUMO to pet-28 - Expression problems with pet SUMO (Jan/05/2010 )

Hi,

I have a gene (from Bacillus thuringiensis) cloned on pET SUMO and we tried to express it on BL21 without success.

Now, we would like to try pET 28a.

How is the best way to transfer my gene from pet sumo to pet 28?

I found that enzymes HindIII, SalI, NheI and NdeI, on pET28a mcs, not cut my gene.

HindIII, NheI and NdeI cut pET SUMO. The first one cleaves downstream gene, on pET SUMO position 659. The others two enzymes cleaves at position 355 and 296, respectively, upstream of my gene.

So, I think that a cleavage with NheI (or NdeI) and HindIII will match with pET28 mcs site. But, I am not sure if the resultant insert will be in frame on pET 28a.

Could you advise me if the resultant pET 28a plasmid harboring a in frame insert if I do that strategies?

Thank you very much!

-tatui-

tatui on Jan 5 2010, 09:21 AM said:

I see you are looking for a shortcut to pull out the gene from pET SUMO! If you want to express from a full ORF, i suggest you re-design primers and introduce appropriate restriction sites that are in pET28a (For directional cloning). I clone it first in pGEMT-Easy, then subclone into pET28a
I hope this helps!

-bargul-

bargul on Jan 5 2010, 03:51 PM said:

I see you are looking for a shortcut to pull out the gene from pET SUMO!

Yes! I figured out that there are enzymes for that.

I am not sure if the cloned gene (plus the pET SUMO upstrem end) will be in frame into pET 28a.

Any suggestion? Is my gene in frame if I do that short cut?

Thank you very much!

-tatui-