Difference between revisions of "Part:BBa K5301001"
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<p>We also employed AlphaFold to predict the structure of the protein constituted by SdyTag, spNW15, and SdyCatcher, and discovered that the SdyTag was successfully bound to SdyCatcher(Figure 1). </p> | <p>We also employed AlphaFold to predict the structure of the protein constituted by SdyTag, spNW15, and SdyCatcher, and discovered that the SdyTag was successfully bound to SdyCatcher(Figure 1). </p> | ||
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| − | + | Figure 1.The structure of the protein constituted by SdyTag, spNW15 and SdyCatcher as predicted by AlphaFold | |
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===Characterization=== | ===Characterization=== | ||
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We used SDS-PAGE to test whether the protein containing SdyCatcher had been expressed successfully(Figure 2). The molecular weight of SCSdC-mCh[1-10] (containing SdyCatcher) is 76.3 kDa. And we purified the target protein with a molecular weight of approximately 70-100 kD (Lane 4 - Lane 5), which demonstrated that we had successfully expressed the protein containing SdyCatcher. | We used SDS-PAGE to test whether the protein containing SdyCatcher had been expressed successfully(Figure 2). The molecular weight of SCSdC-mCh[1-10] (containing SdyCatcher) is 76.3 kDa. And we purified the target protein with a molecular weight of approximately 70-100 kD (Lane 4 - Lane 5), which demonstrated that we had successfully expressed the protein containing SdyCatcher. | ||
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https://static.igem.wiki/teams/5301/parts/sds-page-result-of-sdycatcher-for-sdycatcher.png | https://static.igem.wiki/teams/5301/parts/sds-page-result-of-sdycatcher-for-sdycatcher.png | ||
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Figure 2.SDS-PAGE analysis of the extraction results of SCSdC-mCh[1-10] (containing SdyCatcher).The gel was run at 80 V for 10 minutes and then at 150 V for 20 minutes, followed by staining with Coomassie Brilliant Blue. The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa. | Figure 2.SDS-PAGE analysis of the extraction results of SCSdC-mCh[1-10] (containing SdyCatcher).The gel was run at 80 V for 10 minutes and then at 150 V for 20 minutes, followed by staining with Coomassie Brilliant Blue. The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa. | ||
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Latest revision as of 09:36, 1 October 2024
SdyCatcher can achieve covalent binding of proteins through Tag-Catcher interaction.
SdyCatcher is a fibronectin-binding protein. It can cooperate with SdyTag to achieve covalent binding of proteins through Tag-Catcher interaction. SdyCatcher and SdyTag can also be used alongside SpyTag-SpyCatcher partners, to demonstrate kinetically controlled directed protein ligation, domain specific protein circularization and macromolecular assembly [1].
Usage and Biology
We devised the fusion expression of SdyCatcher with spNW15, SpyCatcher and mCherry[1-10] (namely SCSdC-mCh[1-10]). We employed SdyTag and SdyCatcher to link two proteins - SCSdC-mCh[1-10] and SnCSdT.
We also employed AlphaFold to predict the structure of the protein constituted by SdyTag, spNW15, and SdyCatcher, and discovered that the SdyTag was successfully bound to SdyCatcher(Figure 1).
Figure 1.The structure of the protein constituted by SdyTag, spNW15 and SdyCatcher as predicted by AlphaFold
Characterization
We used SDS-PAGE to test whether the protein containing SdyCatcher had been expressed successfully(Figure 2). The molecular weight of SCSdC-mCh[1-10] (containing SdyCatcher) is 76.3 kDa. And we purified the target protein with a molecular weight of approximately 70-100 kD (Lane 4 - Lane 5), which demonstrated that we had successfully expressed the protein containing SdyCatcher.
Figure 2.SDS-PAGE analysis of the extraction results of SCSdC-mCh[1-10] (containing SdyCatcher).The gel was run at 80 V for 10 minutes and then at 150 V for 20 minutes, followed by staining with Coomassie Brilliant Blue. The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 10
Illegal AgeI site found at 58 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 55
Illegal BsaI.rc site found at 256
- ↑ Tan, L.L., S.S. Hoon and F.T. Wong, Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLOS ONE, 2016. 11(10): p. e0165074.