Difference between revisions of "Part:BBa K5301004"
 
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SpyCatcher comes from the spontaneous isopeptide bond domain in streptococcus pyogenes fibronectin-binding protein FbaB. It can cooperate with SpyTag to achieve covalent binding of proteins through Tag-Catcher interaction. The robust reaction conditions and irreversible linkage of SpyTag-Catcher provide a targetable lock in cells and a stable module for new protein architectures.
 
SpyCatcher comes from the spontaneous isopeptide bond domain in streptococcus pyogenes fibronectin-binding protein FbaB. It can cooperate with SpyTag to achieve covalent binding of proteins through Tag-Catcher interaction. The robust reaction conditions and irreversible linkage of SpyTag-Catcher provide a targetable lock in cells and a stable module for new protein architectures.
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<center><html><img src='https://static.igem.wiki/teams/5301/parts/spytag-nw50-spycatcher.png' width='400px'></html></center>
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<center><html>Figure 1.Structure of Spycatcher-NW50-SpyTag protein extract.From this structure diagram, it is predicted that spytag and spycatcher can be successfully combined at both ends of the spNW50.
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
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<partinfo>BBa_K5301004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5301004 SequenceAndFeatures</partinfo>
  
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<h2>Characterization</h2>
 
<h2>Characterization</h2>
  
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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.
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In the design, we intend to use the robust reaction conditions and irreversible connections of SpyCatcher and SpyTag to link the two ends of the MSP protein, thereby enabling the formation of nanodiscs.
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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 1). A more specific exploration of protein dimerization conditions can be found in the engineering section of iGEM24_BNU-China.
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In the experiment, we successfully characterized the NW50 [BBa_K5301015] protein with spytag and spycatcher, but their presence promoted the dimerization of the protein (Figure 2a). Through the engineering of iGEM24_BNU-China, we alleviated the dimerization problem and produced a monomeric protein with two tags (Figure 2bc).
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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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                <b>Figure 2. SDS analysis of spNW50 with Spycatcher results of dimerization(a) and monomerization(bc).</b>
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<h2>Conclusion</h2>
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Finally, through electron microscope imaging, we determined that the nanodisc was successfully generated under the action of spytag and spycatcher(Figure 3).
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                <b>Figure 3. Electron microscopic images(a) and DLS particle size results(b) of nanodiscs by spNW50 and Spycatcher. </b>
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Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 4). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully.
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                <b>Figure 4. The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone. </b>
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Latest revision as of 04:18, 2 October 2024


SpyCatcher can achieve covalent binding of proteins through Tag-Catcher interaction.

SpyCatcher comes from the spontaneous isopeptide bond domain in streptococcus pyogenes fibronectin-binding protein FbaB. It can cooperate with SpyTag to achieve covalent binding of proteins through Tag-Catcher interaction. The robust reaction conditions and irreversible linkage of SpyTag-Catcher provide a targetable lock in cells and a stable module for new protein architectures.

Figure 1.Structure of Spycatcher-NW50-SpyTag protein extract.From this structure diagram, it is predicted that spytag and spycatcher can be successfully combined at both ends of the spNW50.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

In the design, we intend to use the robust reaction conditions and irreversible connections of SpyCatcher and SpyTag to link the two ends of the MSP protein, thereby enabling the formation of nanodiscs. In the experiment, we successfully characterized the NW50 [BBa_K5301015] protein with spytag and spycatcher, but their presence promoted the dimerization of the protein (Figure 2a). Through the engineering of iGEM24_BNU-China, we alleviated the dimerization problem and produced a monomeric protein with two tags (Figure 2bc).
Figure 2. SDS analysis of spNW50 with Spycatcher results of dimerization(a) and monomerization(bc).

Conclusion

Finally, through electron microscope imaging, we determined that the nanodisc was successfully generated under the action of spytag and spycatcher(Figure 3).
Figure 3. Electron microscopic images(a) and DLS particle size results(b) of nanodiscs by spNW50 and Spycatcher.
Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 4). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully.
Figure 4. The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.