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. Compared with the mother liquor without IPTG induction in the first column, 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 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.

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.