Part:BBa_K5301022

SpyCatcher-spNW50-SpyTag achieving self-cyclization of MSP nanodisc.

This pathway achieving self-cyclization of NW50 to construct nanodiscs with larger particle sizes.

Usage and Biology

The following figure shows the structure of the composite part(Figure 1).

           <a href="" class="image">

<img alt="" src="" width="100%" height=auto class="thumbimage" /></a>

                   <a href="" class="internal" title="Enlarge"></a>

               Figure 1.Structure of Spycatcher-NW50-SpyTag protein extract.From this structure diagram, it is predicted that spytag and spycatcher can be successfully combined at both ends of the NW50. 
               

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1645
  Illegal SpeI site found at 517
  Illegal PstI site found at 733
  Illegal PstI site found at 1273
  Illegal PstI site found at 1306
  Illegal PstI site found at 1813
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1645
  Illegal NheI site found at 133
  Illegal NheI site found at 2061
  Illegal SpeI site found at 517
  Illegal PstI site found at 733
  Illegal PstI site found at 1273
  Illegal PstI site found at 1306
  Illegal PstI site found at 1813
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1645
  Illegal BglII site found at 993
  Illegal BglII site found at 1533
  Illegal BglII site found at 1701
  Illegal BamHI site found at 166
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1645
  Illegal SpeI site found at 517
  Illegal PstI site found at 733
  Illegal PstI site found at 1273
  Illegal PstI site found at 1306
  Illegal PstI site found at 1813
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1645
  Illegal SpeI site found at 517
  Illegal PstI site found at 733
  Illegal PstI site found at 1273
  Illegal PstI site found at 1306
  Illegal PstI site found at 1813
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The total molecular weight of the composite component of Spycatcher-NW50-SpyTag is about 150kDa, and it can be seen that it can be expressed normally after IPTG induction (Figure 2).

Figure 2. SDS analysis of Spycatcher-NW50-SpyTag 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 and the combined action of spytag-spycatcher, Spycatcher-NW50-SpyTag 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 3). A more specific exploration of protein dimerization conditions can be found in the engineering section of iGEM24_BNU-China.

Figure 3. SDS analysis of Spycatcher-NW50-SpyTag results of dimerization(a) and monomerization(bc).

Conclusion

According to the results of electron microscopy and DLS, Spycatcher-NW50-SpyTag and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 200-300nm(Figure 4).

Figure 4. Electron microscopy (a) and DLS results (b) of nanodiscs produced by Spycatcher-NW50-SpyTag. 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.