SpyTag and SpyCatcher are peptides which can associate via a spontaneous amide bond. Because of this, they can be fused to proteins of interest as tags to cause the proteins to bind. There are (currently) three versions of the spytag-spycatcher system, where each system contains a different version of spytag and catcher (i.e. system 1 contains spytag1 and spycatcher1, system2 contains spytag2 and spycatcher2, and system3 contains spytag3 and spycatcher3).


My question is, how compatible are the spytag and spycatchers between different versions? So, for example, would spytag1 bind with spycatcher2?

Prior Research

I found the following statement from this paper:

The new variants showed backwards compatibility, reacting efficiently with parental versions (SpyTag002 with SpyCatcher: 1.0±0.06×104  m−1 s−1; SpyTag with SpyCatcher002: 5.5±0.03×103  m −1 s−1;..

But these numbers don't mean a lot to me. Would you expect to see near complete binding as with spytag2-spycatcher2?


2 Answers 2


I have worked with the coupling of SpyCatchers and SpyTags of different iterations and can confirm that they are all backward compatible. While SpyCatcher003 and SpyTag003 pairs have the quickest reaction rate, reactions of all pairs (including SpyCatcher and SpyTag version 1) should go to completion under the right conditions.

The SpyCatcher002-SpyTag1 is no exception. As you quoted in the Angewandte Chemie paper, the rate of reaction is 5.5x103 M−1 s−1. Rate = k[A][B]. This means that 10 μM of SpyCatcher002 and 10 μM SpyTag will react with a t1/2 (50% coupling) of 18 seconds at pH = 7.0. However, 10 μM of SpyCatcher002 and 10 μM SpyTag2 will t1/2 of 5 seconds (rate constant is 2x104 M−1 s−1). If you are reacting 0.1 μM of each, their respective t1/2s are about 8 and 33 minutes, respectively.

To get the reaction to go to completion (>90% coupling), you'll need to wait ~12 times as long as the t1/2 (since every subsequent t1/2 doubles in a second-order reaction). This will, of course, depend on your concentrations. However, when using low concentrations (0.1 μM), complete coupling of SpyCatcher002 to SpyTag002 will take 1.5 hours, whereas SpyCatcher002 to SpyTag will take 7 hours at 25C. SpyCatcher002 to SpyTag1 will take 3x as long to go to completion; therefore, I'd recommend using at least 1-2 μM of what you want completely coupled with an equal or 2-4x excess amount of either Tag or Catcher and run the reaction overnight. Higher concentrations (>=10 μM of both Tag and Catcher) should go to completion in 30min-1hr. When performing coupling reactions for more than a few hours, I'd recommend you perform them at 4C. Loading 10 μL of 1-2 μM of your protein should allow you to see the band shift on an SDS-PAGE gel with Coomassie stain.

In my experience, temperature and pH are important factors when performing coupling. pH > 8.0 tends to reduce the rate of reaction significantly, as does pushing the temperature upwards of 37C (the protein prefers 25C and pH = 6.0). Most of the reactions I perform in the lab are at 4C overnight at pH = 7.4 between SpyCatcher003 and SpyTag1, and coupling is nearly complete. However, others in our lab have worked extensively with SpyCatcher002 and SpyTag1 reactions, which also tend to go to completion under the right conditions. Moreover, sometimes the protein that the SpyTag is fused to will alter the kinetics of the reaction, and short linkers to SpyTag may reduce your coupling efficiency.

While others are certainly more qualified to answer this question, hopefully this gives you enough information to work with!


I am currently working with these tags, so going off experience here. The binding efficiency is not 100% even when the correct versions are mixed e.g. version 2 spytag and version 2 spycatcher. I believe this is the original paper (https://pubs.acs.org/doi/pdf/10.1021/ja910795a) it reported 60 % binding efficiency after an hour (I presume for version 1) and this paper (https://www.pnas.org/content/pnas/109/12/E690.full.pdf) revealed 40 % binding efficiency in the first minute (unclear whether version 1 or 2 but I presume 1). In terms of between versions I can only speak to a mistake I made once by accidentally mixing version 1 spycatcher with version 2 spytag. There was visible binding that could been seen by SDS-PAGE however, it was very faint and required significant amounts of protein to view (~10 mg/ml). I never did any sort of quantitative measurements on binding efficiency as it was a mistake. But from subsequent correctly run experiments, I could see it was significantly lower so I would always use version 2 with 2 and version 3 with 3 etc as they were designed to have maximum binding efficiencies.


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