Difference between revisions of "Part:BBa K5301015"

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	<h2>Characterization</h2>		<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.	+	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.Compared with the mother liquor without IPTG induction, NW50 protein was successfully expressed(Figure 1).
−	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 iGEM24_BNU-China.	+
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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">
		+	<div class="magnify">
		+	<a href="https://static.igem.wiki/teams/5301/parts/before-and-after-induction-of-nw50-iptg.png" class="internal" title="Enlarge"></a>
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		+	<b>Figure 1. SDS analysis of spNW50 protein extract. Compared with the mother liquor without IPTG induction in the first column, it was obvious that proteins with required molecular weight were produced..</b>
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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(Figure 2). A more specific exploration of protein dimerization conditions can be found in the engineering section of iGEM24_BNU-China.
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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>
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−	<b>Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).</b>	+	<b>Figure 2. SDS analysis of NW50 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, NW50 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 100nm(Figure 3).
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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.jpg" class="internal" title="Enlarge"></a>
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−	<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 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>
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Revision as of 12:28, 30 September 2024

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

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.

Sequence and Features

Characterization

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.Compared with the mother liquor without IPTG induction, NW50 protein was successfully expressed(Figure 1). 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 2). A more specific exploration of protein dimerization conditions can be found in the engineering section of iGEM24_BNU-China.

Conclusion

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 3).

References

[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.