Hello all.
I currently have an issue with N-terminal truncated impurities of desired protein. E. coli has been designed to express the desired protein for analysis and the process has been working just fine for a long time.
Recently, there has been a significant increase in impurities of the desired product (confirmed through LC-MS) which turned out to be N-terminal truncations of the protein of interest. I suspect that these are produced due to enzymatic activity and as I have ruled out mutations (but not post translational modification).
I understand that N-terminal truncations can be caused by aminopeptidases, but what baffles me is that it is happening now of all times. Fermentation/recovery/purification has been working like a charm and to the best of my knowledge I did not make any significant change. After I experienced the spike in N-terminal truncated impurities, it has been a continuing trend and not a one-time phenomenon.
I was wondering if anyone may have any ideas about this? Are there conditions that activate aminopeptidase or cause a large generation of aminopeptidase by the cells?
Is this happening on a single construct, or to different constructs for the expression of different proteins? Are you talking only about removal of the N-terminal methionine or are you detecting different N-terminal truncations?
Hi Tim, many aminopeptidases are Zn+2 dependent enzymes. Try making the preparation 1 mM in 1, 10 Phenanthroline (a Zn chelator) and see if that helps.
Thank you for the answer Steingrimur, I will try that method. But FYI I do use EDTA in the culture and this used to be enough to get expected result. I have always been doing the same thing and one day this just started happening. I did spike the main culture with additional metal ions used in the main culture and I did notice a metal-ion dependent increase in the truncated forms.
Alejandro, this is happening to a single construct for the expression of one particular protein. I am detecting N-terminal truncation between Threonine-Proline linkage.
Hi Tim, EDTA is not an efficient chelator of Zn+2. Another good way to bind metal +2 ions is to use Chelex resin.
www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT200.pdf
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