Part:BBa_K5403005

This part is a variation on Spycatcher ([parts.igem.org/Part:BBa_K4766000 BBa_K4766000]). SpyCatcher is a protein that forms a complex with SpyTag, see figure 1. SpyCatcher and Spytag’s specific binding can be used to bring two biochemical moieties together for many applications. In order to make SpyCatcher suitable for maleimide coupling (bio-orthogonal click chemistry), a cysteine was introduced at the N-terminus. This opens the opportunity to selectively couple many functional groups to Spycatcher's N-terminus, such as fluorescent dyes, saccharides or phospholipids. iGEM Eindhoven 2024 used the cysteine on CysSpycatcher to attach DSPE-MAL, a commercially available phospholipid that is functionalized with a maleimide.

Figure 1: complex of SpyCatcher (green) and SpyTag (orange) (Li et al., 2014).

Results

We coupled Spytag-GFP and CysSpycatcher and ran them on an SDS-PAGE gel to verify the successful conjugation of the two proteins, ensuring that the functional domains of Spytag and Spycatcher interact correctly, forming a stable covalent bond.

Aim:

Check if the recombinantly engineered CysSpycatcher and Spytag-GFP are able to form a covalent isopeptide bond.

Methods:

To couple CysSpycatcher with Spytag-GFP, we prepared the proteins in a 1:1 molar ratio. The two components were mixed in a reaction buffer (e.g., PBS or Tris buffer, pH 7.4) and incubated at room temperature to allow the covalent bond formation between Spycatcher and Spytag.

Results Spytag-Spycatcher coupling:

The SDS-PAGE analysis in Figure 46 reveals several key observations. In the lanes containing CysSpycatcher and Spytag-GFP, the expected bands for each individual protein are present. CysSpycatcher appears as a band around 10 kDa, while Spytag-GFP shows up at approximately 30 kDa. In the lanes where the two proteins were mixed, a prominent new band appears around 40 kDa, consistent with the expected molecular weight of the CysSpycatcher-Spytag-GFP conjugate.

However, there is an unexpected excess of unbound Spytag-GFP in the conjugate lanes, with almost no detectable free CysSpycatcher remaining, despite mixing in a 1:1 molar ratio. This suggests that the CysSpycatcher is fully utilized in binding Spytag-GFP, but for some reason, the Spytag-GFP is in excess. One possible explanation for this observation is inaccuracies in protein quantification or loss of CysSpycatcher during sample preparation.

Additionally, a band is observed around 75 kDa, which could represent one CysSpycatcher protein coupled to two Spytag-GFP proteins. This may indicate that CysSpycatcher is binding more than one Spytag-GFP molecule, possibly due to an incorrect molar ratio during mixing or due to multimerization of proteins. This suggests that optimizing the ratio and reaction conditions could improve the specificity and efficiency of the coupling, preventing the formation of higher molecular weight complexes.

Figure 1: SDS-PAGE gel showing successful covalent coupling between CysSpycatcher (~10 kDa) and Spytag-GFP (~30 kDa). The conjugated product (~40 kDa) is visible in the lanes containing the mixture after incubation, confirming the formation of the CysSpycatcher-Spytag-GFP complex.

Conclusion:

Although the coupling reaction and conditions need further optimization, our results demonstrate that CysSpycatcher and Spytag-GFP successfully combine, as evidenced by the appearance of the expected conjugate band on the SDS-PAGE gel. The presence of higher molecular weight complexes and excess Spytag-GFP suggests room for improvement in the reaction setup, but the fundamental Spycatcher-Spytag interaction has been clearly shown.

Results micelle formation

Aim:

Make micelles that are functionalized with Spycatcher as preparation for the post-insertion method.

Methods:

We prepared micelles using only DSPE-Mal due to the unavailability of DSPE, deviating from the typical DSPE ratio of 4:1. To form the micelles, DSPE-Mal was dissolved in methylene chloride, and a lipid film was created by evaporating the solvent under a nitrogen stream. The lipid film was then hydrated with an appropriate buffer and sonicated to produce micelles.

For functionalization, CysSpycatcher was added to the micellar solution and incubated, allowing the CysSpycatcher to covalently attach to the DSPE-Mal micelles. The micellar solution was then filtered using a 50 kDa centrifugal filter to remove any unbound CysSpycatcher. Next, the filtered DSPE-Mal-CysSpycatcher micelles were incubated with Spytag-GFP to facilitate covalent binding between Spycatcher and Spytag. This solution was filtered again with a 50 kDa centrifugal filter to eliminate any unbound Spytag-GFP. To verify successful functionalization, the DSPE-Mal-CysSpycatcher + Spytag-GFP micelles were compared with a negative control, where DSPE-Mal micelles were directly incubated with Spytag-GFP and filtered using a 50 kDa centrifugal filter to remove unbound Spytag-GFP.

The structural properties of the DSPE-Mal micelles and the unfiltered/filtered DSPE-Mal-CysSpycatcher micelles were evaluated using DLS to assess their size distribution. Additionally, the filtered solution containing DSPE-Mal-CysSpycatcher + Spytag-GFP was compared to the DSPE-Mal + Spytag-GFP negative control using fluorescence analysis to confirm successful functionalization and binding of Spytag-GFP to the CysSpycatcher-modified micelles.

Detailed results are given on the results section of the iGEM Eindhoven 2024 wiki.

Conclusion

The DLS and fluorescence data suggest potential for forming DSPE-mal micelles functionalized with Spycatcher, but micelle formation was inconsistent and showed high variability in size and polydispersity. Non-specific binding of Spytag-GFP was also observed. Overall, this exploratory protocol holds promise but requires significant optimization at each step—micelle formation, functionalization, and filtration—to achieve consistent and reliable results for future applications.

References Li, L., Fierer, J. O., Rapoport, T. A., & Howarth, M. (2014). Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. Journal Of Molecular Biology, 426(2), 309–317. https://doi.org/10.1016/j.jmb.2013.10.021