Difference between revisions of "Part:BBa K5301015"

Revision as of 13:35, 30 September 2024 (view source) Caojiaxin (Talk | contribs) ← Older edit		Revision as of 09:24, 1 October 2024 (view source) Zx1028 (Talk | contribs) Newer edit →
Line 3:		Line 3:
	<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.	+	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.
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here
Line 24:		Line 24:
	<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.Compared with the mother liquor without IPTG induction, NW50 protein was successfully expressed(Figure 1).	+	The theoretical molecular weight of spNW50 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, spNW50 protein was successfully expressed(Figure 1).
	<div class="center">		<div class="center">
Line 41:		Line 41:
	</div>		</div>
−	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.	+	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 iGEM24_BNU-China.
	<div class="center">		<div class="center">
Line 51:		Line 51:
	<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 2. 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>
	</div>		</div>
Line 59:		Line 59:
	<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 200-300nm(Figure 3).	+	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).
	<div class="center">		<div class="center">
Line 69:		Line 69:
	<a href="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph-dls-result.png" 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 3. Electron microscopy (a) and DLS results (b) of nanodiscs produced by NW50. It can be seen that the two results are consistent, and the size of the nanodisc is about 200-300nm.</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>
	</div>		</div>

---

Revision as of 09:24, 1 October 2024

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

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.

Sequence and Features

Characterization

The theoretical molecular weight of spNW50 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, 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 iGEM24_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).

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.