Part:BBa_K5301003

SpyTag can achieve covalent binding of proteins through Tag-Catcher interaction.

SpyTag comes from the spontaneous isopeptide bond domain in streptococcus pyogenes fibronectin-binding protein FbaB. It can cooperate with SpyCatcher to achieve covalent binding of proteins through Tag-Catcher interaction. The robust reaction conditions and irreversible linkage of SpyTag-Catcher provide a targetable lock in cells and a stable module for new protein architectures.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

In the design, we intend to use the robust reaction conditions and irreversible connections of SpyCatcher and SpyTag to link the two ends of the MSP protein, thereby enabling the formation of nanodiscs. In the experiment, we successfully characterized the NW50 [BBa_K5301015] protein with spytag and spycatcher, but their presence promoted the dimerization of the protein (Figure 1a). Through the engineering of iGEM24_BNU-China, we alleviated the dimerization problem and produced a monomeric protein with two tags (Figure 1bc).

Figure 1. SDS analysis of NW50 with Spytag results of dimerization(a) and monomerization(bc).

Conclusion

Finally, through electron microscope imaging, we determined that the nanodisc was successfully generated under the action of spytag and spycatcher(Figure 2).

Figure 2. Electron microscopic images(a) and DLS particle size results(b) of nanodiscs by NW50 and Spytag.