Difference between revisions of "Part:BBa K5246031"
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===Experimental characterization===
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===Protein expression===
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<h2><i>Hirschia baltica</i></h2>
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<p>We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.</p>
  
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====Bioinformatic analysis====
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<p>After SDS-PAGE gel analysis, we concluded that we successfully expressed HfsG, HfsH, HfsK, and HfsL proteins from <i>H. baltica</i>. We noticed that HfsL glycosyltransferase was visible in lower quantities compared to the other proteins. This protein expression conditions need to be further investigated and optimized.</p>
  
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Conserved domain database analysis suggests that HfsH is part of the carbohydrate esterase 4 superfamily and polysaccharide deacetylase family. Proteins of this family may catalyze the N- or O- deacetylation of a substrate. Protein BLAST results show high similarity to peptidoglycan N-acetylglucosamine deacetylase and other polysaccharide deacetylases.
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<p><b>HfsH</b> is visible on the left side of the gel (Fig. 1).</p>
  
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Topology analysis with DeepTMHMM and AlphaFold3 structure showed that HfsH is most probably a globular protein located in the cytoplasm. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions.
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<html>
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<head>
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  <style>
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    .container {
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      display: flex;
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      justify-content: center;
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      align-items: flex-start;
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      gap: 5px; /* Space between table and figure */
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<body>
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  <div class="container">
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    <!-- Table on the left -->
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    <div class="table-container">
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      <h3>Table 1. <i>H. baltica</i> protein sizes in kDa</h3>
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      <table border="1">
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        <tr>
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          <th>Protein Name</th>
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          <th>Size (kDa)</th>
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        </tr>
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        <tr>
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          <td>HfsG</td>
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          <td>37</td>
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        </tr>
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        <tr>
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          <td>HfsH</td>
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          <td>29</td>
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        </tr>
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        <tr>
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          <td>HfsJ</td>
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          <td>41</td>
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        </tr>
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        <tr>
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          <td>HfsK</td>
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          <td>28</td>
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        </tr>
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        <tr>
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          <td>HfsL</td>
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          <td>36</td>
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        </tr>
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      </table>
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    </div>
  
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HfsH is a globular polysaccharide deacetylase that catalyses the deacetylation of N-acetylglucosamine in the holdfast synthesis pathway, previous research supports our conclusions. [1][2][3]
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    <div class="figure-container">
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          <img src="https://static.igem.wiki/teams/5246/results/protein-expression/baltica-expressions.webp" style="width:500px;">
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        </div>
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        <figcaption><center><b>Fig. 1.</b> 12% SDS-PAGE analysis of <i>H. baltica</i> in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific). </center></figcaption>
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    </div>
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  </div>
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</body>
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</html>
  
 
===References===
 
===References===

Revision as of 19:10, 1 October 2024


HB HfsH Deacetylase, 6xHis tag for purification

Introduction

Usage and Biology

TBA

This part also has a non his-tagged variant BBa_K5246020.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 385
  • 1000
    COMPATIBLE WITH RFC[1000]


Protein expression

Hirschia baltica

We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.

After SDS-PAGE gel analysis, we concluded that we successfully expressed HfsG, HfsH, HfsK, and HfsL proteins from H. baltica. We noticed that HfsL glycosyltransferase was visible in lower quantities compared to the other proteins. This protein expression conditions need to be further investigated and optimized.

HfsH is visible on the left side of the gel (Fig. 1).

Table 1. H. baltica protein sizes in kDa

Protein Name Size (kDa)
HfsG 37
HfsH 29
HfsJ 41
HfsK 28
HfsL 36
Fig. 1. 12% SDS-PAGE analysis of H. baltica in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

References

1. Wan, Z. et al. (2013a) ‘The adhesive and cohesive properties of a bacterial polysaccharide adhesin are modulated by a deacetylase’, Molecular Microbiology, 88(3), pp. 486–500. doi:10.1111/mmi.12199.
2. Toh, E., Kurtz, Harry D. and Brun, Y.V. (2008) ‘Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps’, Journal of Bacteriology, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.
3. Liu, Q. et al. (2022) ‘The screening and expression of polysaccharide deacetylase from caulobacter crescentus and its function analysis’, Biotechnology and Applied Biochemistry, 70(2), pp. 688–696. doi:10.1002/bab.2390.