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NusA tag produces protein truncations?

termination truncation nusa solubility

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#1 darel_am

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Posted 16 August 2013 - 02:34 AM

Hi,

 

I am expressing a ~45 kDa fungal protein in E. coli however with a simple his-tag it proved to be insoluble. I then put it into the pET43 and pET44 vectors to include a solublising Nus tag and it is now soluble and I have purified using the his-tag.

 

However I get lots of lower molecular weight bands in this purification and no amount of washing or gradient elution improves this. I have had the lower MW bands identified and they have come back with hits to my protein. So I thought maybe they are truncated protein products.

 

The DNA sequence has been optimised and I have expressed in Rosetta cells with the same banding pattern so it is not a codon issue. I know E. coli struggles to produce proteins > 100 kDa and with the tags my fusion protein is ~110 kDa but I have also just come across the fact that the Nus tag is 491 amino acids of the 495 amino acid NusA protein. I knew NusA was a transcription factor but have just read that one of its purposes is to regulate transcrition termination.

 

My question is, Is the Nus tag active as NusA? Could the overexpression of NusA by means of the Nus tag be acting on my own fusion protein and causing premature termination? I can't find anything that suggests anyone else has encountered this problem, nor can I find what NusA actually terminates!

 

Any comments would be greatly appreciated.

 

Thanks

 

Darel



#2 littlebull

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Posted 19 August 2013 - 08:53 AM

I have used this vector when I was in a lab as a postdoc. As you mentioned, NusA can increase the solubility of your target protein. The cleavage may come from proteases. You try protease inhibitors to avoid cleavage. 



#3 darel_am

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Posted 20 August 2013 - 01:09 AM

I add a protease inhibitor cocktail to my cell lysate so I don't think there is protease action occuring.



#4 littlebull

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Posted 22 August 2013 - 11:44 AM

I still think it is a protease cleavage problem. It may happen at the expression stage. You can take a sample every one hour for SDS-PAGE gel to see when you can see the multibands. You may also have a smaple after breaking cells. Some times, protease inhibitor cocktail may not work well. Do you purify your protein in the cold room? 







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