Thanks a lot for responses. As a summary, I would like to list some typical replies as below:
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Indeed most of the newly synthesized proteins loose their initiator methionine while the protein synthesis is still underway! Initiator methionine is removed by an enzyme called methionine aminopeptidase (MetAP). The only condition is the second residue should be small and uncharged (Gly, Ala, Ser).
Just to introduce to these MetAPs, please look at these references.
A. Addlagatta et al, 2005, Biochemistry, 44, 14741-14749.
A. Addlagatta et al, 2005, Biochemistry, 44, 7166-7174.
Anthony
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The Met was cut off during expresstion. I have a similar situation as
you. Later on, I checked some literature. The efficiency of the
cleavage is dependent on the size of the second amino acid. The smaller
the second amino acid, the higher efficiency. In the case of Ala and
Gly, 99% of the Met got cleaved.
Hope this is helpful,
Ying
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Yes, it happens and depends on sequence.
P.H. Hirel, J.-M. Schmitter, P. Dessen, G. Fayat and S. Blanquet, Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl Acad. Sci. USA 86 (1989), pp. 8247–8251.
Dominika
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In our high-throughput lab, we experience
loss of the N-terminal methionine all the time
E.Coli has a specific peptidase, which removes
this residue of many proteins; From my own
experience, it happens in 30-40% of all
proteins expressed in E.Coli.
Hope this is useful.
Best wishes,
Christoph
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I'd like to echo Matt on the removal of N-terminus Met expressed in E. coli. For eukaryotic (esp. mammalian) proteins it is almost certain that the first Met is removed. I observed with ESIMS that about half of an yeast protein bear the first Met. With my limited experience, both E. coli and Salmonella proteins may retain their first Met in many cases. I am not sure if this removal complicates crystallization.
Yong
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On Sep 26, 2006, at 11:47 AM, Yadong Yu wrote:
Hi,Yadong Yu
This is a ccp4 irrelevant problem but it may be interesting to this group.
Recently I sent 2 freshly purified protein samples for MALDI mass spectra to determine their molecular weights. The results of these 2 protein both were ~135 Da lower than their theoretical molecular weights. It is certainly out of the range of normal error as my proteins have MW less than 20 kDa. The accuracy of these MALDI experiments is reliable, as evidenced by the exact 2-fold ratios between the single-charge value and double-charge value. The expression vector of these 2 proteins were sequenced. They were expressed in Rosetta(DE3), an E.coli strain, and purified in native conditions. Taken account of the size of this difference, it would not be cleavage of some small group, e.g. amino group of Gln/Asn, from certain residues, the more possible reason would be deprivation of amino acid at either end of the protein. In this secenario, the initial methionine may be lost because its peptide fragment (-H2O), of 131 Da in size, matches the difference very well.
Anyone experienced such problem? Is the head of protein already cut during expression, or just lost in MALDI experiments?
Yadong Yu
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LCMN/NICHD/NIH
Bldg 35, Room 3B 1006
35 Lincoln Drive
Bethesda MD 20892
(301)496-9347
Fax (301)496-2396
[EMAIL PROTECTED]
