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<center> <b> Fig. 1. </b> AlphaFold 3 structure showing  </center>
 
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===Protein Expression===
 
===Protein Expression===

Revision as of 18:29, 28 September 2024


CB2/CB2A HfsL Glycosyltransferase, 6xHis tag for purification

Introduction

Usage and Biology

Caulobacter crescentus is a common freshwater gram-negative oligotrophic bacterium of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, C. crescentus daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure holdfast, allowing them to adhere to surfaces and perform cell division.

Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface.

The C. crescentus holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in Escherichia coli.

The holdfast synthesis (hfs) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway.

HfsL in particular is responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule.


This part also has a non 6xhis-tagged variant BBa_K5246012.

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 265
    Illegal NgoMIV site found at 408
    Illegal NgoMIV site found at 639
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

Bioinformatic analysis

CDD analysis showed specific hits in glycosyl transferase family 2. This diverse family transfers sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-abequose to a range of substrates. Protein BLAST further supports these findings and suggests that HfsL is most likely a family 2 glycosyltransferase, which has a domain very similar to the poly-beta-1,6-N-acetyl-D-glucosamine synthase domain of biofilm PGA synthase.

DeepTMHMM analysis suggests that the protein is likely globular and positioned on the inner side of the cell membrane. The AlphaFold3 structure provides additional evidence supporting its globular shape. 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 (Fig.1).

To sum up, HfsL is most probably a globular family 2 glycosyltransferase, responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule, as is further verified by existing research. [1][2][3]

hfsl.png
Fig. 1. AlphaFold 3 structure showing


Protein Expression

   We chose 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 proceeded with conditions optimized in earlier experiments for whole pathway expression: a temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.

   After SDS-PAGE gel analysis, we concluded that HfsG, HfsH, HfsJ, HfsK, HfsL proteins from C. crescentus CB2 and CB2A strains were successfully expressed (Fig.2). We noticed that the glycosyltransferases HfsJ and HfsL were present in lower quantities compared to the other proteins. The expression conditions of both proteins require further investigation and optimization.

   <figure>
       <img src="https://static.igem.wiki/teams/5246/results/protein-expression/cb2-expression.webp" alt="Protein Expression Gel Image">
       <figcaption>Fig.2. 12% SDS-PAGE analysis of C. crescentus CB2 strain proteins in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3h at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).</figcaption>
   </figure>

Protein Sizes

<thead> </thead> <tbody> </tbody>
Protein Name Size (kDa)
HfsG 34
HfsH 27.9
HfsJ 34.7
HfsK 43.3
HfsL 33.3

Protein Purification

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 10mM Tris pH 7.4, 150mM NaCl, and 10mM, 75mM, and 500mM imidazole, respectively. HfsL was successfully purified (fig.3).

<a href="https://static.igem.wiki/teams/5246/results/protein-expression/cb2-l-purification.webp" target="_blank"> <img src="https://static.igem.wiki/teams/5246/results/protein-expression/cb2-l-purification.webp" alt="Protein Purification" width="400"></a>

<figcaption> Fig. 3. 12% SDS-PAGE analysis. C. crescentus CB2 HfsL protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~33 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.</figcaption>

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References

1. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.
2. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.
3. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.