ISISBio:Protocols/Sortase mediated ligation

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, to carry out protein ligation, and to attach proteins to solid supports. 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) in ligation reactions. Although this slows the reaction down significantly it leads to high yields of modified protein with very little hydrolysis product. 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.

We have used the standard Sortase buffer described by ### 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 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 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

• Ligation of small molecules (including fluorophores) to proteins
• Ligation of proteins to solid supports
• Ligation of proteins to DNA oligonucleotides

References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

Contact

• • Cameron Neylon 11:39, 28 May 2008 (EDT)