Part:BBa_K5301013

spNW15

Introduction

The goal of BNU-China 2024 iGEM team is to fabricate nanodiscs, a kind of engineered nanoscale tool, by means of synthetic biology. Our parts collection can be mainly divided into two categories: mono-MSPs that could construct small or large nanodiscs through self-cyclization, and large cyclic MSP formed by the interaction and linkage of multiple MSPs, which are used for constructing large nanodiscs. They are closely linked together due to their common function of manufacturing nanodiscs.

Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs. We further sought and obtained spNW15 and spNW50 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. Only when three mono-MSPs interact with each other can they form cyclized MSP and achieve their function of constructing nanodiscs. It provides 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.

This part produces spNW15, manufacturing small-diameter and easily cyclized nanodiscs, and can be flexibly modified to produce larger nanodiscs.

Usage and Biology

SpNW15 is the truncated form of spNW30. spNW15 could construct nanodiscs with diameter of 15nm, and is circularized by SpyTag-Spycatcher.

Characterization

The theoretical molecular weight of NW15 is 40.3kDa, 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. According to the theroetical weight of spNW15, it was obvious that proteins with required molecular weight were produced.

After SDS-PAGE, we performed size exclusion chromatograghy on the proteins with the aforementioned concentrations of 100 mM and 300 mM imidazole, and obtained the pure protein(figure 2)

Figure 2. SDS analysis of size exclusion chromatograghy flow through. According to the theroetical weight of spNW15, it was obvious that we had obtain the pure protein.

Conclusion

According to the results of electron microscopy , NW15 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 10nm(Figure 3).

Figure 3. The results of electron microscopy

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]