Part:BBa_K5301024
Multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MS
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[1]. We further sought and obtained spNW15 and spNW50 [2]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[3].
This part is the multi-polymerized MSP which is a large-sized cyclized MSP that connects three MSP parts through different linkers, facilitating the manufacture of larger nanodiscs.
Usage and Biology
In order to produce large nanodiscs more conveniently, we hope to flexibly extend the length of MSP according to demand, and thus propose the concept of multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MSP fragments. We used NW15 as the basic MSP and selected three types of linkers (Spy/Sdy/Snoop) to achieve the connection of different MSP fragments through the formation of covalent bonds, and adopted rigorous design to prevent self-cyclization of each fragment of the multi-polymer MSP. Finally, the successful cyclization of large circular MSPs is characterized by the fluorescence of mCherry after the combination of mCherry [1-10] and mCherry [11].
We also utilized AlphaFold 2 to simulate the overall structure of multi-polymerized MSP, and obtained multi-polymerized MSP with the correct conformation, as shown in the figure.
Figure 1.Alpha Fold 2 prediction model of the overall structure of multi-polymerized MSP
Characterization
We attempted to mix the three components of multi-polymerized MSP in vitro under two different conditions: first, by centrifuging the bacterial cultures, resuspending them, mixing the three bacterial suspensions, and then sonicating the mixture, followed by incubating the protein mixture at 4°C, additionally, the obtained mixture was concentrated to make the target bands more prominent; alternatively, by mixing the three highly purified proteins after purification through nickel affinity chromatography. We verified the efficiency of the assembly of the three components through SDS-PAGE, as shown in the figure below.
From the figure, it can be observed that compared to the monomeric MSPs, the mixed product exhibits two new bands at approximately 100 and 180 kDa. These are presumed to be multi-polymerized MSP.
Figure 2.SDS-PAGE analysis of the construction of multi-polymerized MSP.
Structure and biological activity analysis
Since the results from SDS-PAGE did not provide us with reliable data, we aimed to characterize the successful construction of the multi-polymerized MSP through the observation of mCherry fluorescence. We used a fluorescence inverted microscope to observe the mixture obtained from the first method mentioned above and found red fluorescence under green light excitation, thus confirming the successful ligation of the mCherry protein. Based on this, we could indicate that we have successfully constructed the multi-polymerized MSP.
Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3141
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 3141
Illegal NotI site found at 1720
Illegal NotI site found at 4038 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1239
Illegal BglII site found at 2323
Illegal BglII site found at 2695
Illegal BglII site found at 3557
Illegal BglII site found at 3929 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3141
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3141
Illegal AgeI site found at 1035
Illegal AgeI site found at 1828
Illegal AgeI site found at 2220
Illegal AgeI site found at 2980 - 1000COMPATIBLE WITH RFC[1000]
- ↑ Ilia G. Denisov, Bradley J. Baas, Yelena V. Grinkova, Stephen G. Sligar, Cooperativity in Cytochrome P450 3A4: LINKAGES IN SUBSTRATE BINDING, SPIN STATE, UNCOUPLING, AND PRODUCT FORMATION*, Journal of Biological Chemistry, Volume 282, Issue 10, 2007, Pages 7066-7076, ISSN 0021-9258, https://doi.org/10.1074/jbc.M609589200.
- ↑ 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.
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