Protein/protein-crosslinkers

Deitiker, Philip R via methods%40net.bio.net (by pdeitik from bcm.tmc.edu)
Wed Nov 21 14:16:51 EST 2007


 

The anhydride bond is one of the most difficult to form. 

In peptide synthesis the anhydride that is used to react with the amino
terminal of the growing peptide is formed by carbodiimide. The anhydride of 
carbonyl groups is unstable and will be attacked by the lysine amino groups causing the permanent formation R-CO-N-R bond. 

Typically dicyclohexyl carbodiimide or diisopropyl carbodiimide. 
There are water soluble carbodiimied such as 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimidic hydrochloride. 
Pierce cat.# 22980. More crossreactivity can be achieved by making a succinic
anhydride of the protein of interest which has a higher crosslinking activity
to targets. The target sites are Lysines, amino termini. 

This is not suitable for treatment of living cells. Carbodiimides are known as sensitizing agents, they do crosslink proteins on the skin of laboratory researches which can link gliadin (such as used to be used as a donning agent) latex and self proteins. Individuals develop dermatitis to the new
antigens and this then can cause allergies to gloves, donning agents, etc. 

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

There is an enzyme in living cells known as transglutaminase (tTG),
it can be found in the nucleus, cytosol, and extracellular matrix. 

http://en.wikipedia.org/wiki/Tissue_transglutaminase
http://en.wikipedia.org/wiki/Keratinocyte_transglutaminase

tTG crosslinks 4 amino acid motives (for a discussion of targets
review literature on gliadin as these have been the most characterized)
It crosslinks the lysine amino group to glutamines and to a lessor degree apsartamine.
Guinea pig transglutaminase is available, to be used the target needs to have a 4 amino acid motif that is identified by the enzyme and the enzyme will cross link other proteins to it. This will likely cause ubiquitization and targeting for degradation. 

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Peter Henriksen
Sent: Wednesday, November 21, 2007 9:41 AM
To: methods from magpie.bio.indiana.edu
Subject: Protein/protein-crosslinkers

I am trying to design an experiment in which I will need to make protein-protein crosslinks but not DNA-protein crosslinks in living cells. 
 
In principal I think this should be possible using a crosslinker that will crosslink between two carboxylic groups but I haven't been able to find such a crosslinker so far.

 
If anyone has knowledge of a crosslinker with the above properties I would be thrilled to know about it. 





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