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C-terminal Cleavage During Overexpression


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#1 connoc4

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Posted 25 January 2011 - 01:40 PM

Hi,

I'm currently using E. coli to express a fusion protein, but my fusion protein keeps getting cleaved on the C-terminus at various points. I know this because I can overexpress and purify the isotopically-labeled fusion proteins, separate the fusion proteins of various lengths with HPLC, and then use NMR to see that the C-terminal residues are missing. The solubility tag I'm using works so well that I can't get the fusion protein to express into the inclusion body to try and prevent cleavage.


Does anybody know if there is something that I can add to my cultures to prevent C-terminal cleavage? I'm trying to avoid making a new fusion protein construct.


Thanks!!!!

#2 rbrgouda

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Posted 23 February 2011 - 06:50 AM

Hi,

I'm currently using E. coli to express a fusion protein, but my fusion protein keeps getting cleaved on the C-terminus at various points. I know this because I can overexpress and purify the isotopically-labeled fusion proteins, separate the fusion proteins of various lengths with HPLC, and then use NMR to see that the C-terminal residues are missing. The solubility tag I'm using works so well that I can't get the fusion protein to express into the inclusion body to try and prevent cleavage.


Does anybody know if there is something that I can add to my cultures to prevent C-terminal cleavage? I'm trying to avoid making a new fusion protein construct.


Thanks!!!!



#3 rbrgouda

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Posted 23 February 2011 - 06:51 AM

Hi,
I notice similar problem with my protein. I am over-expressing this protein in E.coli with a N-terminal SUMO-tag (12 kDa). The cleavage is happening near N-terminal end of protein of interest (not inside the SUMO-tag region). As seen on the gel, about equal amounts of full-length and truncated version (N-terminal cleaved) are produced at the time of over-expression. I never tried expressing the protein in to inclusion body. Always I wanted to express it into soluble fraction. Let me know if you find any solution.

thanks
-rbrgouda

#4 mdfenko

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Posted 23 February 2011 - 08:56 AM

you may try a carboxypeptidase inhibitor in your medium when you induce your protein.
talent does what it can
genius does what it must
i do what i get paid to do




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