ISISBio:Protocols/Sortase mediated ligation/Protein DNA ligation
The preparation of proteins carrying a specific piece of DNA is desirable for a range of applications in high sensitivity analysis, the preparation of protein arrays, and other applications in analysis and, potentially, nanotechnology. Sortase mediated ligation provides an attractive approach to this modification through the preparation of glycine modified oligonucleotides. This work is more preliminary than what we have done in other areas but we describe it here so that others can use it. As the oligonucleotides are generally expensive it is difficult to provide them in a large excess. The yields are lower in any case. We have generally found that using a relatively low concentration of target protein (10 µM) and a 5-10 fold excess of oligo gives acceptable results (~50% yield based on protein)
Design and preparation of modified oligonucleotide
DNA is both much larger than the small molecules we have ligated to proteins and also highly charged. There is also the potential for the glycine amino group to form an internal ionic reaction with the phosphate backbone, reducing its availability for ligation. We have carried out a partial optimisation of the number of glycines attached to the 5’ end of an oligonucleotide. This remains preliminary at this stage.
Constructs of the form H-Gn-Sp-oligo-OH where Sp is a spacer (generally aminooctanoic acid or a similar amino PEG) can be formed in two ways. A phosporamidite of the form Fmoc-Gn-Sp-P can be synthetically prepared and coupled as part of the DNA synthesis. We have prepared the Fmoc protected single glycine phosphoramidite with an aminooctanoic acid spacer. The Fmoc-G-Aoc intermediate in this synthesis is very poorly soluble in the solvents used for phosphoramidite preparation and the phosphoramidite is only sparingly soluble in solvents used for DNA synthesis. Our attempts to prepare the equivalent diglycine phosphoramidite were unsuccessful as we could not dissolve the protected diglycine-spacer intermediate in any useful solvent. This may be easier if a PEG or other more soluble spacer is used.
The preferred approach to preparation of the glycine modified oligo is therefore post synthesis coupling of the Fmoc protected amino acid or peptide. This is most conveniently carried out on the column after gentle deprotection of a 5’ amino terminated phosphoramidite spacer. Fmoc-diglycine can be readily prepared by standard methods. Fmoc-tetraglycine is again highly insoluble and the preparation of tetraglycine modified oligonucleotides will probably require two coupling steps.
We have attempted ligations using oligonucleotides modified with one glycine (prepared via the phosphoramidite method) and two glycines (via the coupling method). Both yield product that migrates slower by SDS-PAGE and faster by HPLC on a C4 column. Yields for the single glycine modified oligo are poor whereas the optimised yield for the diglycine modified oligo are around 50-70% based on the amount of target protein. Clearly it would be desirable to test tetraglycine modified oligonucleotides and we are currently pursuing their preparation. Specialist oligonucletide companies (those that prepare high quality and complex labelled oligonucleotides, such as ATDBio) should be able to prepare diglycine modified oligonucleotides to order. They are not likely to be cheap, however the price may well come down if this becomes a popular modification.
- Sortase A
- LPETGG-tagged protein target
- Gycine modified oligonucleotide
- Sortase buffer: 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.5
Standard Ligation Conditions
- Target protein: 5 – 10 µM
- Glycine modified oligonucleotide: 20 - 100 µM (5-10 fold excess over protein depending on how much oligo is available)
- Sortase A: 50 nM
- Mix reactants together and incubate overnight at room temperature.
- Run gel to confirm labeling reaction
- Purify product
The product should generally be clear by SDS-PAGE as a slower migrating band (see here-lanes 5 and 7 for an example of SDS-PAGE analysis). We have not attempted the purification of product but this should be feasible using a combination of gel filtration and/or anion exchange chromatography.
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 would probably make it possible to ligate an aminolink oligonucleotide with low yield and low purity as well as speeding 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.
Relevant papers and books
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- Cameron Neylon 11:45, 28 May 2008 (EDT)