ISISBio:Protocols/Sortase mediated ligation

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==Introduction==
==Introduction==
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The Sortase A enzyme of S. aureus provides an effective means of ligating a range of molecules to the C-termini of proteins. Sortase can be used to ligate small molecules including fluorophores to protein <cite>popp07</cite>, to carry out protein and polymer ligation <cite>mao04 parthasarathy07 </cite>, and to attach proteins to solid supports <cite>parthasarathy07 chan07</cite>. The major considerations in such experiments are the design of the protein tag for modification and the construction of the ligation partner. In these protocols we discuss the use of Sortase to modify proteins with small molecule fluorophores, to attach proteins to solid supports, and to ligate proteins to oligonucleotides.
+
The Sortase A enzyme of S. aureus provides an effective means of ligating a range of molecules to the C-termini of proteins. Sortase can be used to ligate small molecules including fluorophores to protein <cite>popp07 neylon07</cite>, to carry out protein and polymer ligation <cite>mao04 parthasarathy07 </cite>, and to attach proteins to solid supports <cite>parthasarathy07 chan07</cite>. The major considerations in such experiments are the design of the protein tag for modification and the construction of the ligation partner. In these protocols we discuss the use of Sortase to modify proteins with small molecule fluorophores, to attach proteins to solid supports, and to ligate proteins to oligonucleotides.
==General considerations==
==General considerations==
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Sortase mediated ligation is an equilibrium process with the products generally being potential reactants. To improve the yield of product it is therefore advisable to provide a significant excess of the ligation partner over the target protein. A large excess leads in most cases to quantitative yields of modified protein. Using an excess of ligation partner can lead to problems in purification after the ligation but we have generally found that it is more straightforward to purify modified protein from excess ligation partner than to attempt the purification of modified from unmodified protein. We generally aim for 100-fold excess in small molecule ligation reactions.
Sortase mediated ligation is an equilibrium process with the products generally being potential reactants. To improve the yield of product it is therefore advisable to provide a significant excess of the ligation partner over the target protein. A large excess leads in most cases to quantitative yields of modified protein. Using an excess of ligation partner can lead to problems in purification after the ligation but we have generally found that it is more straightforward to purify modified protein from excess ligation partner than to attempt the purification of modified from unmodified protein. We generally aim for 100-fold excess in small molecule ligation reactions.
-
In addition there is a potential side product of the reaction, the’shunt’ or hydrolysis product of attack on the enzyme bound thioester intermediate by water.  The yield of this side product is increased by having a low concentration of ligation partner but is most directly effected by the concentration of enzyme thioester intermediate in the reaction. This in turn is directly related to the concentration of enzyme in the reaction. Therefore, in contrast to other reports, we advocate the use of low concentrations of Sortase (~50 nM) in ligation reactions. Other groups have generally used high concentrations of Sortase (50 μM) <cite>parthasarathy07 popp07 samantaray08</cite> and observe ligation to alkylamines <cite>parthasarathy07 samantaray 08</cite> at much higher levels than we do. Although using lower Sortase concentrations slows the reaction down significantly it leads to high yields of modified protein with very little hydrolysis product in our hands. As the hydrolysis product is usually very difficult to purify from the desired ligation product we believe that on balance a degree of patience is advised.
+
In addition there is a potential side product of the reaction, the’shunt’ or hydrolysis product of attack on the enzyme bound thioester intermediate by water.  The yield of this side product is increased by having a low concentration of ligation partner but is most directly effected by the concentration of enzyme thioester intermediate in the reaction. This in turn is directly related to the concentration of enzyme in the reaction. Therefore, in contrast to other reports, we advocate the use of low concentrations of Sortase (~50 nM) where possible in ligation reactions. Other groups have generally used high concentrations of Sortase (50 μM) <cite>parthasarathy07 popp07 samantaray08</cite> and observe ligation to alkylamines <cite>parthasarathy07 samantaray 08</cite> at much higher levels than we do. Although using lower Sortase concentrations slows the reaction down significantly it leads to high yields of modified protein with very little hydrolysis product in our hands. As the hydrolysis product is usually very difficult to purify from the desired ligation product we believe that on balance a degree of patience is advised. However we have also noted significant batch to batch variation of Sortase activity and activity against different substrates. In all cases thus far we have been able to get good yields of ligation by increasing the Sortase concentration to ~1 uM but we are currently not sure what the source of variation is. This is currently under investigation and we will be re-cloning and completely re-sequencing the Sortase gene in our plasmid pLLC064.
-
We have used the standard Sortase buffer described by ### for ligation reactions and have not investigated varying it to any great extent. The CaCl<sub>2</sub> is described as increasing the rate of reaction. A number of reports have discussed optimal pH and other operating conditions for the enzyme.
+
We have used the standard Sortase buffer described by Ton-That et al. <cite>ton-that00</cite> for ligation reactions and have not investigated varying it to any great extent. The CaCl<sub>2</sub> is described as increasing the rate of reaction. A number of reports have discussed optimal pH and other operating conditions for the enzyme.
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These are the general reagents used for all ligation reactions. Discussion of the specific reagents will be in each individual protocol.
These are the general reagents used for all ligation reactions. Discussion of the specific reagents will be in each individual protocol.
-
Sortase: A Sortase  A construct with the N-terminal membrane targeting sequence removed is readily purified in N-terminally His-tagged form. The pETMCSIII based vector pLLC064 is available for expression of this construct. The protein is easily overexpresed and purified using standard nickel affinity chromatography conditions. It should be noted that the protein migrates anomalously on SDS-PAGE gels and appears to run as a protein of ~26 kDa molecular weight (###). We have confirmed the correct molecular weight of Sortase A expressed from pLLC064 by MS. The protein is very stable. We find it convenient to store the protein in Sortase buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl<sub>2</sub>, pH 7.5). The protein appears stable to multiple freeze-thaw cycles. We have not as yet investigated the potential to freeze dry Sortase for storage although we hope to carry out this investigation soon.
+
Sortase: A Sortase  A construct with the N-terminal membrane targeting sequence removed is readily purified in N-terminally His-tagged form. The pETMCSIII based vector pLLC064 is available for expression of this construct. The protein is easily overexpresed and purified using standard nickel affinity chromatography conditions. It should be noted that the protein migrates anomalously on SDS-PAGE gels and appears to run anomalouslyas a protein of higher molecular weight (see also <cite>ton-that99</cite> figure 5). We have confirmed the correct molecular weight of Sortase A expressed from pLLC064 by MS. We generally use a lower concentration of Sortase (50 nM) than reported by others but have seen significant batch to batch variation in activity and activity against different targets. Under most circumstances poor yields appear to be overcome by increasing Sortase concentrations to 1 - 10 μM but this has the disadvantage of potential side reactions such as hydrolysis and contamination of products with large quantities of protein. We find it convenient to store the protein in Sortase buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl<sub>2</sub>, pH 7.5). The protein appears stable to multiple freeze-thaw cycles. We have not as yet investigated the potential to freeze dry Sortase for storage although we hope to carry out this investigation soon.
Protein target: We have used a range of proteins as targets for Sortase mediated ligation including a set of fluorescent proteins, the redox protein flavadoxin reductase, and the DNA binding protein Tus. In every case we have observed essentially quantitative ligation yields under optimal conditions (large excess of ligation partner) at room temperature. We have not systematically investigated the role of spacers or flexibility in the yield of reaction. In general we have placed a three amino acid spacer  (usually GSG) followed by two amino acids encoded by the XhoI site we use in our cloning strategy, followed by the Sortase A recognition sequence LPETGG. In our construct this is followed by a His<sub>6</sub> tag. This provides both a convenient initial purification strategy and a means of partially purifying ligated from unreacted (but not from hydrolysed) protein.
Protein target: We have used a range of proteins as targets for Sortase mediated ligation including a set of fluorescent proteins, the redox protein flavadoxin reductase, and the DNA binding protein Tus. In every case we have observed essentially quantitative ligation yields under optimal conditions (large excess of ligation partner) at room temperature. We have not systematically investigated the role of spacers or flexibility in the yield of reaction. In general we have placed a three amino acid spacer  (usually GSG) followed by two amino acids encoded by the XhoI site we use in our cloning strategy, followed by the Sortase A recognition sequence LPETGG. In our construct this is followed by a His<sub>6</sub> tag. This provides both a convenient initial purification strategy and a means of partially purifying ligated from unreacted (but not from hydrolysed) protein.
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==Contact==
==Contact==
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**'''[[User:Cameron Neylon|Cameron Neylon]] 11:39, 28 May 2008 (EDT)'''
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*'''[[User:Cameron Neylon|Cameron Neylon]] 11:39, 28 May 2008 (EDT)'''
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Current revision

Contents

Introduction

The Sortase A enzyme of S. aureus provides an effective means of ligating a range of molecules to the C-termini of proteins. Sortase can be used to ligate small molecules including fluorophores to protein [1, 2], to carry out protein and polymer ligation [3, 4], and to attach proteins to solid supports [4, 5]. The major considerations in such experiments are the design of the protein tag for modification and the construction of the ligation partner. In these protocols we discuss the use of Sortase to modify proteins with small molecule fluorophores, to attach proteins to solid supports, and to ligate proteins to oligonucleotides.

General considerations

Sortase mediated ligation is an equilibrium process with the products generally being potential reactants. To improve the yield of product it is therefore advisable to provide a significant excess of the ligation partner over the target protein. A large excess leads in most cases to quantitative yields of modified protein. Using an excess of ligation partner can lead to problems in purification after the ligation but we have generally found that it is more straightforward to purify modified protein from excess ligation partner than to attempt the purification of modified from unmodified protein. We generally aim for 100-fold excess in small molecule ligation reactions.

In addition there is a potential side product of the reaction, the’shunt’ or hydrolysis product of attack on the enzyme bound thioester intermediate by water. The yield of this side product is increased by having a low concentration of ligation partner but is most directly effected by the concentration of enzyme thioester intermediate in the reaction. This in turn is directly related to the concentration of enzyme in the reaction. Therefore, in contrast to other reports, we advocate the use of low concentrations of Sortase (~50 nM) where possible in ligation reactions. Other groups have generally used high concentrations of Sortase (50 μM) [1, 4, 6] and observe ligation to alkylamines [4, 7, 8] at much higher levels than we do. Although using lower Sortase concentrations slows the reaction down significantly it leads to high yields of modified protein with very little hydrolysis product in our hands. As the hydrolysis product is usually very difficult to purify from the desired ligation product we believe that on balance a degree of patience is advised. However we have also noted significant batch to batch variation of Sortase activity and activity against different substrates. In all cases thus far we have been able to get good yields of ligation by increasing the Sortase concentration to ~1 uM but we are currently not sure what the source of variation is. This is currently under investigation and we will be re-cloning and completely re-sequencing the Sortase gene in our plasmid pLLC064.

We have used the standard Sortase buffer described by Ton-That et al. [9] for ligation reactions and have not investigated varying it to any great extent. The CaCl2 is described as increasing the rate of reaction. A number of reports have discussed optimal pH and other operating conditions for the enzyme.


Materials

These are the general reagents used for all ligation reactions. Discussion of the specific reagents will be in each individual protocol.

Sortase: A Sortase A construct with the N-terminal membrane targeting sequence removed is readily purified in N-terminally His-tagged form. The pETMCSIII based vector pLLC064 is available for expression of this construct. The protein is easily overexpresed and purified using standard nickel affinity chromatography conditions. It should be noted that the protein migrates anomalously on SDS-PAGE gels and appears to run anomalouslyas a protein of higher molecular weight (see also [10] figure 5). We have confirmed the correct molecular weight of Sortase A expressed from pLLC064 by MS. We generally use a lower concentration of Sortase (50 nM) than reported by others but have seen significant batch to batch variation in activity and activity against different targets. Under most circumstances poor yields appear to be overcome by increasing Sortase concentrations to 1 - 10 μM but this has the disadvantage of potential side reactions such as hydrolysis and contamination of products with large quantities of protein. We find it convenient to store the protein in Sortase buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The protein appears stable to multiple freeze-thaw cycles. We have not as yet investigated the potential to freeze dry Sortase for storage although we hope to carry out this investigation soon.

Protein target: We have used a range of proteins as targets for Sortase mediated ligation including a set of fluorescent proteins, the redox protein flavadoxin reductase, and the DNA binding protein Tus. In every case we have observed essentially quantitative ligation yields under optimal conditions (large excess of ligation partner) at room temperature. We have not systematically investigated the role of spacers or flexibility in the yield of reaction. In general we have placed a three amino acid spacer (usually GSG) followed by two amino acids encoded by the XhoI site we use in our cloning strategy, followed by the Sortase A recognition sequence LPETGG. In our construct this is followed by a His6 tag. This provides both a convenient initial purification strategy and a means of partially purifying ligated from unreacted (but not from hydrolysed) protein.

Sortase Buffer:50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.5

Specific Protocols

References

Relevant papers and books

  1. Popp MW, Antos JM, Grotenbreg GM, Spooner E, and Ploegh HL. . pmid:17891153. PubMed HubMed [popp07]
  2. Cameron Neylon, Chemtools LaBLog, 29 August 2007 http://chemtools.chem.soton.ac.uk/projects/blog/blogs.php/bit_id/2709 [neylon07]
  3. Mao H, Hart SA, Schink A, and Pollok BA. . pmid:14995162. PubMed HubMed [mao04]
  4. Parthasarathy R, Subramanian S, and Boder ET. . pmid:17302384. PubMed HubMed [parthasarathy07]
  5. Chan L, Cross HF, She JK, Cavalli G, Martins HF, and Neylon C. . pmid:18000537. PubMed HubMed [chan07]
  6. Samantaray S, Marathe U, Dasgupta S, Nandicoori VK, and Roy RP. . pmid:18229923. PubMed HubMed [samantaray08]
  7. Ton-That H, Mazmanian SK, Faull KF, and Schneewind O. . pmid:10734144. PubMed HubMed [ton-that00]
  8. Ton-That H, Liu G, Mazmanian SK, Faull KF, and Schneewind O. . pmid:10535938. PubMed HubMed [ton-that99]
All Medline abstracts: PubMed HubMed

Contact

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