Hello all,

I expressed my fusion protein bound to MBP using a pMAL-c2e vector and purified it using an amylose column. Upon running a cleavage reaction of 1 mg of my fusion protein in 1 mL of 20 IU of enterokinase for 12h at 25 C. I ran it in a 4 mL 30k MWCO centrifugal spin filter at 500xg for 2h aspirating every 30 min. However, I could not get any of the target protein that's 21kDa into the filtrate upon seeing the gel of it. The gel depicted is what was in the concentrate and not the filtrate as there was nothing in it.

I'm reckon I used too much enterokinase as I will have to redo the pilot cleavage reaction with lower amounts, but that's a lack of foresight on my part. Alternatively, I could also use a weak anion exchanger like DEAE instead.

What do you guys think?

Thanks,

Ostin