Difference between revisions of "Part:BBa K5301015"

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	<partinfo>BBa_K5301015 short</partinfo>		<partinfo>BBa_K5301015 short</partinfo>
−	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.	+	==Introduction==
−	<!-- Add more about the biology of this part here	+	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.
−	===Usage and Biology===	+	<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>
		+	<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>
−	<!-- -->	+	<p>This part produces spNW50, manufacturing large-diameter and easily cyclized nanodiscs.</p>
−	===Sequence and Features===	+	==Usage and biology==
−	<span class='h3bb'></span>	+
−	<partinfo>BBa_K5301015 SequenceAndFeatures</partinfo>	+
		+	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.
−	<!-- Uncomment this to enable Functional Parameter display	+	==Characterization==
−	===Functional Parameters===	+
−	<partinfo>BBa_K5301015 parameters</partinfo>	+	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">
		+	<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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		+	<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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−	<h2>Characterization</h2>
−	The theoretical molecular weight of NW50 is 123.9kDa[1], and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is 150kDa.	+	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.
−	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(Figure 1). A more specific exploration of protein dimerization conditions can be found in the engineering section of 2024BNUChina iGEM.	+
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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>		<a href="https://static.igem.wiki/teams/5301/parts/sds-results-of-nw50-dimerization-or-monomerization.png" class="internal" title="Enlarge"></a>
	</div>		</div>
−	<b>Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).</b>	+	<b>Figure 2. SDS analysis of spNW50 results of dimerization(a) and monomerization(bc).</b>
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	<h2>Conclusion</h2>		<h2>Conclusion</h2>
−	According to the results of electron microscopy and DLS, NW50 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 100nm(Figure 2).	+	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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−	<a href="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph.jpg" class="image">	+	<a href="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph-dls-result.png" class="image">
−	<img alt="" src="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph.jpg" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">	+	<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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−	<a href="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph.jpg" class="internal" title="Enlarge"></a>	+	<a href="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph-dls-result.png" class="internal" title="Enlarge"></a>
	</div>		</div>
−	<b>Figure 2. Electron microscopic images of nanodiscs with a particle size of about 100nm prepared by NW50. The particle size is in agreement with our DLS results.</b>	+	<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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		+	</html>
		+	<!-- Add more about the biology of this part here
−	<h2>References</h2>	+	<!-- -->
−	[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.	+
		+	===Sequence and Features===
		+	<span class='h3bb'></span>
		+	<partinfo>BBa_K5301015 SequenceAndFeatures</partinfo>
		+
		+
		+	<!-- Uncomment this to enable Functional Parameter display
		+	===Functional Parameters===
		+	<partinfo>BBa_K5301015 parameters</partinfo>
		+	<!-- -->

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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).

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.

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).

Sequence and Features

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.