Part:BBa_K5301015

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

Introduction

The goal of the BNU-China 2024 iGEM team is to fabricate nanodiscs, a kind of engineered nanoscale tool, by means of synthetic biology. Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs. We further sought and obtained spNW15 and spNW50 ^[1]that utilized the automatic covalent linkage of SpyTag and SpyCatcher to enhance the cyclization efficiency and enable the automatic cyclization of MSP, in order to manufacture nanodiscs of different diameters more simply. On this basis, taking NW15 as the basic component, we designed the multi-polymerized MSP, consisting of three linear MSP monomers, providing a more flexible solution for manufacturing large nanodiscs, while reducing the expression pressure on the chassis bacteria and avoiding the difficulty of purifying large proteins.

This Part Collection aims to provide a series of easily accessible and distinctively characterized MSP proteins as a toolkit for the assembly of nanodiscs. Users can easily select which MSP to produce and utilize based on their own needs to manufacture nanodiscs. The nanodiscs fabricated using the MSP we designed can be used for stabilizing amphipathic proteins, studying the structure and function of amphipathic proteins, drug delivery, developing novel antiviral drugs, etc., and possess broad application prospects^[2].

This part produces spNW50, manufacturing large-diameter and easily cyclized nanodiscs.

Usage and biology

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.

Characterization

The theoretical molecular weight of spNW50 is 123.9kDa^[3], 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 second 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 iGEM 2024 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.

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]

↑ Zhang, S., et al., One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 2021. 12(1): p. 5451.
↑ Padmanabha Das, K.M., et al., Large Nanodiscs: A Potential Game Changer in Structural Biology of Membrane Protein Complexes and Virus Entry. Frontiers in Bioengineering and Biotechnology, 2020. 8.
↑ 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.

[1] Zhang, S., et al., One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 2021. 12(1): p. 5451.

[2] Padmanabha Das, K.M., et al., Large Nanodiscs: A Potential Game Changer in Structural Biology of Membrane Protein Complexes and Virus Entry. Frontiers in Bioengineering and Biotechnology, 2020. 8.

[3] 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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