ISISBio:Protocols/Sortase mediated ligation/Small molecule ligation

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==Overview==
==Overview==
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We have used Sortase to attach small molecule fluorophores to proteins but there is no reason why similar constructs shouldn't work for a very wide range of small molecule labels. Obvious examples include azides, biotin, and cysteine. We generally carry the reaction out overnight at room temperature and have not examined the effect of temperature extensively.
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We <cite>neylon07</cite> and others <cite>popp07</cite> 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) <cite>samantaray08</cite> 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==
==Design of the small molecule ligation partner==
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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.
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We have investigated the rate and yield of reaction for substrates of the form H-GG<sub>n</sub>K(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==
==Materials==
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*LPETGG-tagged protein target
*LPETGG-tagged protein target
*Small molecule construct
*Small molecule construct
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*Sortase buffer
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*Sortase buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl<sub>2</sub>, pH 7.5)
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==Standard Ligation Conditions==
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*Target protein: 5 – 200 µM
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*Ligation partner: 100 µM – 1 mM (at least 20-fold excess over protein)
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*Sortase A: 50 nM (although sometimes poor yields can be overcome by higher concentrations see the [[ISISBio:Protocols/Sortase_mediated_ligation|main Sortase page]] for details).
==Procedure==
==Procedure==
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#Mix reactants together and incubate overnight at room temperature. Yes, it really is that simple (see ### for an example).
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#Mix reactants together and incubate overnight at room temperature. Yes, it really is that simple (see [http://chemtools.chem.soton.ac.uk/projects/blog/blogs.php/bit_id/2709 here] for an example).
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#Run gel to confirm labeling reaction if appropriate
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#Purify product
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'''Relevant papers and books'''
'''Relevant papers and books'''
<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
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<biblio>
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<blockquote>
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#Goldbeter-PNAS-1981 pmid=6947258
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<biblio force=false>
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#Jacob-JMB-1961 pmid=13718526
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#Ptashne-Genetic-Switch isbn=0879697164
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#[[ISISBio:Protocols/Sortase_mediated_ligation/References]]
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</biblio>
</biblio>
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</blockquote>
==Contact==
==Contact==
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**'''[[User:Cameron Neylon|Cameron Neylon]] 11:45, 28 May 2008 (EDT)''':Who has experience with this protocol?
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**'''[[User:Cameron Neylon|Cameron Neylon]] 11:45, 28 May 2008 (EDT)'''
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[[Category:In vitro]]
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[[Category:Protein]]
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[[Category:Chemical]]
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[[Category:Labeling]]
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Current revision

Contents

Overview

We [1] and others [2] 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) [3] 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


Notes

The small molecule ligation partner can be readily removed by gel filtration and residual Sortase can be removed if required by gel filtration (if the sizes are sufficiently different) or nickel affinity chromatography. Remember the desired product is in the flow through.

The yield of desired product generally follows the ratio of protein to ligation partner. Reducing the concentration of ligation partner will therefore reduce the yield. It also tends to slightly increase the amount of hydrolysis product observed. Increasing the Sortase concentration will speed up the reaction but will also increase the amount of hydrolysis product. Reactions are probably complete in 4-6 hours depending on target protein concentration but we have found overnight incubation convenient.

References

Relevant papers and books

  1. Cameron Neylon, Chemtools LaBLog, 29 August 2007 http://chemtools.chem.soton.ac.uk/projects/blog/blogs.php/bit_id/2709 [neylon07]
  2. Popp MW, Antos JM, Grotenbreg GM, Spooner E, and Ploegh HL. . pmid:17891153. PubMed HubMed [popp07]
  3. Samantaray S, Marathe U, Dasgupta S, Nandicoori VK, and Roy RP. . pmid:18229923. PubMed HubMed [samantaray08]
All Medline abstracts: PubMed HubMed

Contact

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