Part:BBa_K5301013

spNW15

SpNW15 is the truncated form of spNW30. spNW15 could construct nanodiscs with diameter of 15nm, and is circularized by SpyTag-Spycatcher.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 496
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 496
  Illegal NheI site found at 64
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 496
  Illegal BglII site found at 552
  Illegal BglII site found at 924
  Illegal BamHI site found at 97
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 496
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 496
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  Illegal AgeI site found at 1027
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The theoretical molecular weight of NW15 is 40.3kDa[1], and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is around 40kDa. spNW15 protein was successfully expressed(Figure 1).

Figure 1. SDS analysis of spNW15 protein extract. According to the theroetical weight of spNW15, it was obvious that proteins with required molecular weight were produced.

After SDS-PAGE, we performed Size-Exclusion Chromatography on the proteins with the aforementioned concentrations of 100 mM and 300 mM imidazole, and obtained the pure protein(figure 2)

Figure 2. SDS analysis of Size-Exclusion Chromatography flow through. According to the theroetical weight of spNW15, it was obvious that we had obtain the pure protein.

Conclusion

According to the results of electron microscopy , NW15 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 10nm(Figure 3).

Figure 2. The results of electron microscopy

References

[1]Zhang, S., Ren, Q., Novick, S.J. et al. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nat Commun 12, 5451 (2021). https://doi.org/10.1038/s41467-021-25737-7.