Part:BBa_K5301015

spNW50 is high molecular weight membrane scaffold protein used to produce large nanodiscs.

spNW50 is high molecular weight membrane scaffold protein used to produce large nanodiscs.spNW50 could construct nanodiscs with large diameter, and is circularized by SpyTag-Spycatcher.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 496
  Illegal EcoRI site found at 1576
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  Illegal PstI site found at 1204
  Illegal PstI site found at 1237
  Illegal PstI site found at 1744
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 496
  Illegal EcoRI site found at 1576
  Illegal NheI site found at 64
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  Illegal PstI site found at 1204
  Illegal PstI site found at 1237
  Illegal PstI site found at 1744
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 496
  Illegal EcoRI site found at 1576
  Illegal BglII site found at 924
  Illegal BglII site found at 1464
  Illegal BglII site found at 1632
  Illegal BamHI site found at 97
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 496
  Illegal EcoRI site found at 1576
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  Illegal PstI site found at 1204
  Illegal PstI site found at 1237
  Illegal PstI site found at 1744
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 496
  Illegal EcoRI site found at 1576
  Illegal SpeI site found at 448
  Illegal PstI site found at 664
  Illegal PstI site found at 1204
  Illegal PstI site found at 1237
  Illegal PstI site found at 1744
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The theoretical molecular weight of spNW50 is 123.9kDa[1], and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is 150kDa.Compared with the mother liquor without IPTG induction, spNW50 protein was successfully expressed(Figure 1).

Figure 1. SDS analysis of spNW50 protein extract. Compared with the mother liquor without IPTG induction in the first column, it was obvious that proteins with required molecular weight were produced.

Due to the large molecular weight of the protein, spNW50 is prone to dimerization during actual protein extraction. Protein dimerization can be minimized by adding the detergent Triton X-100 to the refined extraction buffer and reducing the nickel column elution time during the refined extraction process. It should be used as soon as possible after protein extraction to prevent the protein from self-dimerizing after a period of time(Figure 2). A more specific exploration of protein dimerization conditions can be found in the engineering section of iGEM24_BNU-China.

Figure 2. SDS analysis of spNW50 results of dimerization(a) and monomerization(bc).

Conclusion

According to the results of electron microscopy and DLS, spNW50 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 200-300nm(Figure 3).

Figure 3. Electron microscopy (a) and DLS results (b) of nanodiscs produced by spNW50. It can be seen that the two results are consistent, and the size of the nanodisc is about 200-300nm.

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.