Re: [ccp4bb] E. coli mutant strains
Le 26/02/2011 03:51, Dima Klenchin a écrit : It surely is not. An N-end rule has to do with ubiquitination, and it is absent in E.coli. Not true. There is indeed and N-end rule in prokaryotes, including E. coli. Mediated by the ClpP protease-based system. See: http://www.cell.com/molecular-cell/abstract/S1097-2765%2808%2900692-8 -- Miguel Architecture et Fonction des Macromolécules Biologiques (UMR6098) CNRS, Universités d'Aix-Marseille I II Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France Tel: +33(0) 491 82 55 93 Fax: +33(0) 491 26 67 20 mailto:miguel.ortiz-lombar...@afmb.univ-mrs.fr http://www.afmb.univ-mrs.fr/Miguel-Ortiz-Lombardia -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] E. coli mutant strains
It surely is not. An N-end rule has to do with ubiquitination, and it is absent in E.coli. Not true. There is indeed and N-end rule in prokaryotes, including E. coli. Mediated by the ClpP protease-based system. See: http://www.cell.com/molecular-cell/abstract/S1097-2765%2808%2900692-8 Ooops! I dodn't know that. Live and learn. Thanks, Miguel! Dima
Re: [ccp4bb] E. coli mutant strains
If you want a ClpP minus strain you can get it from the Keio strains. http://cgsc.biology.yale.edu/index.php http://www.shigen.nig.ac.jp/ecoli/strain/top/top.jsp For expression with the T7 promotor, you will need to use the λDE3 Lysogenization Kit or use a Arabinose induction plasmid instead.
Re: [ccp4bb] E. coli mutant strains
Dear Jerry, Whats about an N-terminal His-Tag with an Xa factor cleavage site behind it... After IMAC you only get protein with the entire N-Terminus (His-Tag), afterward digest the protein with Xa factor...it won¹t leave any additional amino acids C-terminally! You¹ll get your protein! ;) Cheers, Christian Am 2/26/11 2:15 AM schrieb Jerry McCully unter for-crystallizai...@hotmail.com: Dear ALL: Recently I am expressing one protein in BL21(DE3) and the protein undergoes N-terminal degradation. I am trying to keep this crucial N-terminal tail on the protein, which has MRS at the first 3 positions. Digging in to the literatures, I found the N-end rule, which tell that the proteins bearing N-terminal Arg or lys have much shorter half life. I am not sure whether this is my problem. However, I want to found one E. coli mutant strain that lacks aat and is unable to degrade N-end rule substrates that bear N-terminal Arg or Lys. Does anybody know such strains? Thanks a lot and have a nice weekend, Jerry,
Re: [ccp4bb] E. coli mutant strains
Factor Xa will work depending on the exact sequence. If your sequence starts MRS... and the start methionine is important and not removed then yes it will work when you clone it into a Factor Xa site. If however the start sequence starts RS... and the start methionine is actually removed, when you clone the gene minus the start Met, Factor Xa will not cleave IEGR/RS. Have you done Mass spec on the protein to confirm that the start methionine is removed/present? Dan Dear Jerry, Whats about an N-terminal His-Tag with an Xa factor cleavage site behind it... After IMAC you only get protein with the entire N-Terminus (His-Tag), afterward digest the protein with Xa factor...it won’t leave any additional amino acids C-terminally! You’ll get your protein! ;) Cheers, Christian
[ccp4bb] E. coli mutant strains
Dear ALL: Recently I am expressing one protein in BL21(DE3) and the protein undergoes N-terminal degradation. I am trying to keep this crucial N-terminal tail on the protein, which has MRS at the first 3 positions. Digging in to the literatures, I found the N-end rule, which tell that the proteins bearing N-terminal Arg or lys have much shorter half life. I am not sure whether this is my problem. However, I want to found one E. coli mutant strain that lacks aat and is unable to degrade N-end rule substrates that bear N-terminal Arg or Lys. Does anybody know such strains? Thanks a lot and have a nice weekend, Jerry,
Re: [ccp4bb] E. coli mutant strains
Recently I am expressing one protein in BL21(DE3) and the protein undergoes N-terminal degradation. I am trying to keep this crucial N-terminal tail on the protein, which has MRS at the first 3 positions. Digging in to the literatures, I found the N-end rule, which tell that the proteins bearing N-terminal Arg or lys have much shorter half life. I am not sure whether this is my problem. It surely is not. An N-end rule has to do with ubiquitination, and it is absent in E.coli. However, I want to found one E. coli mutant strain that lacks aat and is unable to degrade N-end rule substrates that bear N-terminal Arg or Lys. Don't think such a strain exists and totally not sure this is really your problem. (Also, what's AAT has to do with it? Is this your requirement for something else?) Try inhibiting proteases better. E.g., metal-dependent proteases are a common problem with IMAC-based purification. First thing is to find whether the degradation occurs inside the cells or during purification step(s). If the former, why not fuse the thing to an N-terminal tag, thus only purifying non-degraded N-termini? (Assuming a monomer, of course). If N-term. has to be native, His-tagged SUMO fusion cleaved with SUMO protease will leave no non-native residues (assuming none was introduced during cloning, which is rather trivial to ensure these days). - Dima
