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<partinfo>BBa_K5301022 short</partinfo>
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This pathway achieving self-cyclization of NW50 to construct nanodiscs with larger particle sizes.The following figure shows the structure of the composite part(Figure 1).
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This pathway achieving self-cyclization of spNW50 to construct nanodiscs with larger particle sizes.
<center><html>Figure 1.Structure of Spycatcher-spNW50-SpyTag protein extract.From this structure diagram, it is predicted that spytag and spycatcher can be successfully combined at both ends of the NW50.
<b>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.</b>
SpyCatcher-spNW50-SpyTag achieving self-cyclization of MSP nanodisc.
This pathway achieving self-cyclization of spNW50 to construct nanodiscs with larger particle sizes.
Figure 1.Structure of Spycatcher-spNW50-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.
