Coding
Part:BBa_K5301022
Designed by: Jiaxin Cao Group: iGEM24_BNU-China (2024-09-24)
SpyCatcher-spNW50-SpyTag achieving self-cyclization of MSP nanodisc.
This pathway expresses the SpyCatcher-NW50-SpyTag fusion protein under IPTG induction, achieving self-cyclization of NW50 to construct nanodiscs.
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE 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 1). 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 2). A more specific exploration of protein dimerization conditions can be found in the engineering section of iGEM24_BNU-China.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 3).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.
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Categories
Parameters
None |