Hello everyone, I am currently working with targeting proteins to yeast mitochondria. Our strategy is to use a mitochondrial targeting signal to transport our protein of interest to mitochondrial matrix, after import to mitochondria, the peptide is cleaved. I have successfully obtained the yeast strain with the correct plasmids and very good purification experiments.
I have sequenced the purified protein by Edman degradation and found out that the N-terminal sequence is not exactly as expected. The fist amino acids of the purified protein is: EQKPS. But there is no E in that position, it should be another Q. I have checked the plasmid sequence and it is correct, CAGCAA. Have someone faced the same problem?
Thank you very much in advance, any insight is appreciated.
I cannot say much about the possibilities for something having gone wrong in your molecular biology part, but have a look at cyclization of glu to pyroglutamic acid.
https://en.wikipedia.org/wiki/Pyroglutamic_acid
Hi Achim Recktenwald the pyroglutamic acid was the first thing I found about this issue. But when I contacted the Edman sequencing service, they told me that if that was the case, they shouldn't have seen the E peak in the chromatograph, so in theory that is not the cause for what I am seen in the results. Thank you
Glutamine is notoriously unstable and can undergo deamidation, yielding glutamic acid.
Pierre Béguin , YES, forgot about that. This is the most likely case.
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