Difference between revisions of "Part:BBa K5301015"
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<partinfo>BBa_K5301015 short</partinfo>
 
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NW50 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.
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==Introduction==
  
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====Characterization====
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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.
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The theoretical molecular weight of NW50 is 123.9kDa, and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is 150kDa.
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<p>Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs<ref>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.</ref>. We further sought and obtained spNW15 and spNW50 <ref> Zhang, S., et al., One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 2021. 12(1): p. 5451.</ref>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. </p>
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Due to the large molecular weight of the protein, NW50 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. A more specific exploration of protein dimerization conditions can be found in the engineering section of 2024BNUChina iGEM.
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<p>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<ref> 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.</ref>. </p>
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According to the results of electron microscopy and DLS, NW50 and lipid DOPC can be successfully used to fabricate nanodisks with a particle size of about 100nm(Figure 2).
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph.jpg</div><div class="thumbcaption">Figure 2. Electron microscopic images of nanodisks with a particle size of about 100nm prepared by NW50. The particle size is in agreement with our DLS results.</div></div></div></div>
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<p>This part produces spNW50, manufacturing large-diameter and easily cyclized nanodiscs.</p>
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==Usage and biology==
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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==
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The theoretical molecular weight of spNW50 is 123.9kDa<ref>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.</ref>, 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).
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            <a href="https://static.igem.wiki/teams/5301/parts/before-and-after-induction-of-nw50-iptg.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/5301/parts/before-and-after-induction-of-nw50-iptg.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
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                </div>
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                <b>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.</b>
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            </div>
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        </div>
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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.
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<div class="center">
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    <div class="thumb tnone">
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            <a href="https://static.igem.wiki/teams/5301/parts/sds-results-of-nw50-dimerization-or-monomerization.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/5301/parts/sds-results-of-nw50-dimerization-or-monomerization.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
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                <div class="magnify">
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                    <a href="https://static.igem.wiki/teams/5301/parts/sds-results-of-nw50-dimerization-or-monomerization.png" class="internal" title="Enlarge"></a>
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                </div>
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                <b>Figure 2. SDS analysis of spNW50 results of dimerization(a) and monomerization(bc).</b>
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        </div>
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<h2>Conclusion</h2>
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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).
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<div class="center">
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    <div class="thumb tnone">
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        <div class="thumbinner" style="width:50%;">
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            <a href="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph-dls-result.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph-dls-result.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
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                <div class="magnify">
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                    <a href="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph-dls-result.png" class="internal" title="Enlarge"></a>
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                </div>
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                <b>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.</b>
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
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<partinfo>BBa_K5301015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5301015 SequenceAndFeatures</partinfo>
  

Latest revision as of 10:07, 2 October 2024


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

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 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[4], 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]


  1. ↑ 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.
  2. ↑ Zhang, S., et al., One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 2021. 12(1): p. 5451.
  3. ↑ 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.
  4. ↑ 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.