Difference between revisions of "Part:BBa K5246026"
(→‎Bioinformatic analysis)
Line 29: Line 29:
  
 
HfsG is family 2 glycosyltransferase similar to WecA of E.Coli. This globular protein transfers UDP-GlcNAc to the acceptor molecule, our conclusions are in agreement with existing research. [1][2][3]
 
HfsG is family 2 glycosyltransferase similar to WecA of E.Coli. This globular protein transfers UDP-GlcNAc to the acceptor molecule, our conclusions are in agreement with existing research. [1][2][3]
 +
 +
===Protein purification===
 +
<h2>CB2 strain</h2>
 +
<p>After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.</p>
 +
 +
<p><b>HfsJ</b> protein was tricky to purify; after expression at low levels, it eluded earlier than expected at 75mM imidazole, but with some optimization, it should be well purifyable  .</p>
 +
 +
<html>
 +
</p>
 +
<figure>
 +
<div class = "center" >
 +
<center>
 +
<img src = "https://static.igem.wiki/teams/5246/results/protein-expression/cb2-h-purification.webp" style = "width:300px;"></center>
 +
</div>
 +
 +
<figcaption><center>
 +
<b>Fig. 4.</b> 12% SDS-PAGE analysis. <i>C. crescentus</i> CB2 <b>HfsH</b> protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 28 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.</center></figcaption>
 +
</figure>
 +
<p/>
 +
</html>
 +
 +
<html>
 +
<h2>CB2A strain</h2>
 +
<p>After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.</p>
 +
 +
<p><b>HfsJ</b> Surprisingly, in comparison with CB2 strain protein CB2A was quite well purifyable considering quite low expression levels and is well seen in the elution fraction at expected size of 34.5kDa  .</p>
 +
 +
<html>
 +
</p>
 +
<figure>
 +
<div class = "center" >
 +
<center>
 +
<img src = "https://static.igem.wiki/teams/5246/results/protein-expression/cb2a-h-purification.webp" style = "width:300px;"></center>
 +
</div>
 +
 +
<figcaption><center>
 +
<b>Fig. 5.</b> 12% SDS-PAGE analysis. <i>C. crescentus</i> CB2 <b>HfsH</b> protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 28 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.</center></figcaption>
 +
</figure>
 +
<p/>
 +
</html>
  
 
===References===
 
===References===

Revision as of 09:24, 29 September 2024


CB2/CB2A HfsH Deacetylase, 6xHis tag for purification

Introduction

Usage and Biology

TBA

This part also has a non his-tagged variant BBa_K5246008.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 10
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 10
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 10
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 10
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 10
    Illegal NgoMIV site found at 68
    Illegal NgoMIV site found at 92
    Illegal NgoMIV site found at 96
    Illegal NgoMIV site found at 169
    Illegal NgoMIV site found at 295
    Illegal NgoMIV site found at 456
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

Bioinformatic analysis

CDD and protein BLAST analysis suggest that HfsG is a glucosyltransferase family 2 protein. Proteins of this family are involved in cell wall biosynthesis. HfsG is similar to the WecA protein in E.Coli that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate. In the case of HfsG, it catalyzes the transfer of UDP-GlcNAc to the sugar acceptor made earlier in the holdfast synthesis pathway.

Protein topology analysis using DeepTMHMM suggests that HfsG is a globular protein located in the cytoplasm. AlphaFold 3 structures further confirm it. 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.

HfsG is family 2 glycosyltransferase similar to WecA of E.Coli. This globular protein transfers UDP-GlcNAc to the acceptor molecule, our conclusions are in agreement with existing research. [1][2][3]

Protein purification

CB2 strain

After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.

HfsJ protein was tricky to purify; after expression at low levels, it eluded earlier than expected at 75mM imidazole, but with some optimization, it should be well purifyable .

Fig. 4. 12% SDS-PAGE analysis. C. crescentus CB2 HfsH protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 28 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.

CB2A strain

After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.

HfsJ Surprisingly, in comparison with CB2 strain protein CB2A was quite well purifyable considering quite low expression levels and is well seen in the elution fraction at expected size of 34.5kDa .

Fig. 5. 12% SDS-PAGE analysis. C. crescentus CB2 HfsH protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 28 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.

References

1. 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.
2. Hardy, G.G. et al. (2018) ‘Mutations in sugar-nucleotide synthesis genes restore holdfast polysaccharide anchoring to Caulobacter crescentus holdfast anchor mutants’, Journal of Bacteriology, 200(3). doi:10.1128/jb.00597-17.
3. Sulkowski, N.I. et al. (2019) ‘A multiprotein complex anchors adhesive holdfast at the outer membrane of Caulobacter crescentus’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00112-19.