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Why is my His-SUMO tag being cleaved during purification?

sumo protein purification pet28b fusion protein

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#1 robradford

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Posted 04 February 2019 - 01:32 PM

Hi All,

 

It's been a while since I posted on BioForum!

 

I'm having a problem I didn't anticipate. I'm purifying a protein from E. coli (Rosetta) tagged with 6xHis-SUMO.

 

I have a band roughly where I expect it to be in the lysate, but when I mix with my Ni-NTA beads and elute I have an odd pattern.

 

I get a huge band roughly the size of SUMO, and only a very small band at the expected size. My lysis buffers all have protease inhibitor cocktail added.

 

This is the 1st time I've ever tried to purify protein from bacterial cells so I'm really at a loss as to what my problem is. Any help would be appreciated!

 

All the best,

 

Rob



#2 OldCloner

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Posted 05 February 2019 - 10:51 AM

Hi- I don't have the answer but I assume you are using a vector like pET-SUMO, and the SUMO cleavage is only supposed to happen with a specific protease, right?  If I were you I'd contact the manufacturer of the vector for advice about your purification method and see what they say.  Manufacturers know a lot about their stuff, so give them a call! I've learned a lot that way! good luck!







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