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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 nanodisks. 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).
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Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).
Conclusion
Finally, through electron microscope imaging, we determined that the nanodisk was successfully generated under the action of spytag and spycatcher(Figure 2).
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Figure 2. Electron microscopic images of nanodiscs with a particle size of about 100nm prepared by NW50. The particle size is in agreement with our DLS results.
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