ISISBio:Protocols/Sortase mediated ligation/Small molecule ligation

Overview
We neylon07 and others popp07 have used Sortase to attach small molecules fluorophores to proteins but there is no reason why similar constructs shouldn't work for a very wide range of small molecule labels. Ligation of amino sugars has been demonstrated (using much higher Sortase concentrations than we generally do) samantaray08 and other obvious possibilities include azides, alkynes, alkenes, biotin, and cysteine. We generally carry the reaction out overnight at room temperature and have not examined the effect of temperature extensively.

Design of the small molecule ligation partner
We have investigated the rate and yield of reaction for substrates of the form H-GGnK(Fluor)-OH which are straightforward to synthesise and relatively cheap to purchase from general peptide suppliers. We have observed no difference in rate of reaction or yield for n=1-3 and would therefore advise the use of substrates of the form H-GGK(Fluor)-OH on the grounds of ease and price. We have used fluorescein amine exclusively. Others have reported the use of similar constructs with three glycines and rhodamine but we see no reason why other fluorophores and other molecules should not work with two glycines. We recommend using as high a concentration of ligation partner as is feasible (or affordable) to obtain the highest yield.

Materials

 * Sortase A
 * LPETGG-tagged protein target
 * Small molecule construct
 * Sortase buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.5)

Standard Ligation Conditions

 * Target protein: 5 – 200 µM
 * Ligation partner: 100 µM – 1 mM (at least 20-fold excess over protein)
 * Sortase A: 50 nM (although sometimes poor yields can be overcome by higher concentrations see the main Sortase page for details).

Procedure

 * 1) Mix reactants together and incubate overnight at room temperature. Yes, it really is that simple (see here for an example).
 * 2) Run gel to confirm labeling reaction if appropriate
 * 3) Purify product

Contact

 * Cameron Neylon 11:45, 28 May 2008 (EDT)