Difference between revisions of "Part:BBa K5246027"

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===Introduction===
 
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This protein is part of the Tetrad assembly system <a href="https://parts.igem.org/Part:BBa_K5246043">BBa_K5246043</a> and operon responsible for addition of N-acetyl-D-glucosamine to N-acetyl-D-glucosamine and deacetylation of the said molecule <a href="https://parts.igem.org/Part:BBa_K5246042">BBa_K5246042</a>. </p>
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This protein is part of the Tetrad assembly system <a href="https://parts.igem.org/Part:BBa_K5246043">BBa_K5246043</a> and operon responsible for addition of N-acetyl-D-glucosamine to N-acetyl-D-glucosamine and deacetylation of the said molecule <a href="https://parts.igem.org/Part:BBa_K5246041">BBa_K5246041</a>. </p>
  
 
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This part also has a non 6xhis-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246012">BBa_K5246012</a>.
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This part also has a non 6xhis-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246010">BBa_K5246010</a>.
 
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===Usage and Biology===
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===Biology and usage===
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<h2>Biology</h2>
 
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<b>HfsJ</b> in particular most probably responsible for mannosaminuronic acid transfer to the glucose acceptor molecule.
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<b>HfsJ</b>, in particular, is responsible for manosaminuronic acid transfer to the glucose acceptor molecule.
 
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<h3>HfsJ</h3>
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Gene HfsJ from <i>Caulobacter crescentus</i> encodes a protein of 316aa. HfsJ is one of the glycosyltransferases involved in the holdfast synthesis pathway and is structurally very similar to glycosyltransferases that transfer UDP-N-acetyl-D-mannosaminuronic acid.
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<h2>Usage</h2>
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  Proteins of the holdfast synthesis system assemble a short chain of sugar monomers in a specific sequence on a lipid carrier - a glycolipid.
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  Glycolipids are predominantly located on the extracellular surface of eukaryotic cell membranes and are responsible for various functions such as receptors for viruses and other pathogens, allowing them to enter a specific host cell that has unique glycolipid markers. This feature can let us use said glycolipids as labels for a precise and targeted liposome distribution throughout the body, delivering anything from cancer drugs to gene editing systems directly to the target cells.
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  To create a liposome labeling system, we had to select specific proteins that could be utilized for this purpose. Following bioinformatics analysis using the Conserved Domain Database, Protein BLAST, DeepTMHMM, and AlphaFold 3, we identified five proteins of interest from each strain: HfsG, HfsH, HfsJ, HfsK, and HfsL.
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  To utilize these enzymes, it was essential to develop a suitable purification strategy. For efficient cloning, we chose Golden Gate assembly. For efficient purification, we selected immobilized ion affinity chromatography (IMAC) as our purification method, based on recommendations from one of the few available papers where <i>C. crescentus</i> proteins were expressed and purified from <i>E. coli</i>. We opted for conventional 6x histidine tags (his-tag) to facilitate straightforward purification. It was crucial to determine the appropriate terminus for 6xHis-tag insertion to avoid disrupting the protein conformation and lessening purification efficiency.
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===Sequence and Features===
 
===Sequence and Features===
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===Experimental characterization===
 
  
 
====Bioinformatic analysis====
 
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<center> <b> Fig. 1. </b> AlphaFold 3 structure showing </center>
 
<center> <b> Fig. 1. </b> AlphaFold 3 structure showing </center>
  
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===Experimental characterization===
  
 
===Protein expression===
 
===Protein expression===
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h2>CB2A strain</h2>
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<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>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>
  
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<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>
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<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 34.7kDa .</p>
  
 
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Latest revision as of 13:57, 2 October 2024


CB2/CB2A HfsJ Glycosyltransferase, 6xHis tag for purification


Introduction

Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - C. crescentus and H. Baltica - harness 12 protein synthesis pathways to produce sugars anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used E. coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.


This protein is part of the Tetrad assembly system BBa_K5246043 and operon responsible for addition of N-acetyl-D-glucosamine to N-acetyl-D-glucosamine and deacetylation of the said molecule BBa_K5246041.

Part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/.

This part also has a non 6xhis-tagged variant BBa_K5246010.

Biology and usage

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 called holdfast, allowing them to adhere to surfaces and perform cell division.[1][2]

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[3][4].

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[4][5][6].

HfsJ, in particular, is responsible for manosaminuronic acid transfer to the glucose acceptor molecule.

HfsJ

Gene HfsJ from Caulobacter crescentus encodes a protein of 316aa. HfsJ is one of the glycosyltransferases involved in the holdfast synthesis pathway and is structurally very similar to glycosyltransferases that transfer UDP-N-acetyl-D-mannosaminuronic acid.

Usage

Proteins of the holdfast synthesis system assemble a short chain of sugar monomers in a specific sequence on a lipid carrier - a glycolipid.

Glycolipids are predominantly located on the extracellular surface of eukaryotic cell membranes and are responsible for various functions such as receptors for viruses and other pathogens, allowing them to enter a specific host cell that has unique glycolipid markers. This feature can let us use said glycolipids as labels for a precise and targeted liposome distribution throughout the body, delivering anything from cancer drugs to gene editing systems directly to the target cells.

To create a liposome labeling system, we had to select specific proteins that could be utilized for this purpose. Following bioinformatics analysis using the Conserved Domain Database, Protein BLAST, DeepTMHMM, and AlphaFold 3, we identified five proteins of interest from each strain: HfsG, HfsH, HfsJ, HfsK, and HfsL.

To utilize these enzymes, it was essential to develop a suitable purification strategy. For efficient cloning, we chose Golden Gate assembly. For efficient purification, we selected immobilized ion affinity chromatography (IMAC) as our purification method, based on recommendations from one of the few available papers where C. crescentus proteins were expressed and purified from E. coli. We opted for conventional 6x histidine tags (his-tag) to facilitate straightforward purification. It was crucial to determine the appropriate terminus for 6xHis-tag insertion to avoid disrupting the protein conformation and lessening purification efficiency.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 632
    Illegal BamHI site found at 780
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 353
  • 1000
    COMPATIBLE WITH RFC[1000]


Bioinformatic analysis

CDD analysis revealed that HfsJ is part of the WecB/TgA/CpsF glycosyltransferase family. This family catalyzes the formation of glycosidic bonds and may be involved in the biosynthesis of repeating polysaccharide units found in membrane glycolipids. It has domains very similar to E. coli WecG glycosyltransferase, which is responsible for UDP-N-acetyl-D-mannosaminuronic acid transfer. Results are supported by the protein BLAST, which showed significant similarities with the same WecG glycosyltransferase from E. coli.

DeepTMHMM analysis predicted that HfsJ is a globular protein located on the cytoplasmic side of the membrane.

AlphaFold 3 structure, with a high confidence score, shows that HfsJ is most likely a globular protein mostly made of alpha helices. 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).

Based on our findings and prior research, we propose that HfsJ is likely a globular protein responsible for transferring UDP-N-acetyl-D-mannosaminuronic acid and catalyzing the formation of a glycosidic bond. [7][8]

hfsj.png
Fig. 1. AlphaFold 3 structure showing


Experimental characterization

Protein expression

CB2 strain

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 all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.

HfsJ is visible in the center of the gel (Fig. 2).

Table 1. C. crescentus protein sizes in kDa

Protein Name Size (kDa)
HfsG 34
HfsH 27.9
HfsJ 34.7
HfsK 43.3
HfsL 33.3
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 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

CB2A strain

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 all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.

HfsJ is visible in the center of the gel (Fig. 2).

Table 2. C. crescentus protein sizes in kDa

Protein Name Size (kDa)
HfsG 34
HfsH 27.9
HfsJ 34.7
HfsK 43.3
HfsL 33.3
Fig. 3. 12% SDS-PAGE analysis of C. crescentus CB2A strain proteins 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).

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 HfsJ protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 34.7 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 34.7kDa .

Fig. 5. 12% SDS-PAGE analysis. C. crescentus CB2 HfsJ protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 34.7 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. Hendrickson, H., & Lawrence, J. G. (2000). Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. FEMS Microbiology Reviews, 24(2), 177–183. https://doi.org/10.1111/j.1574-6976.2000.tb00539.x
2. Andrews, S. C., Robinson, A. K., & Rodríguez-Quiñones, F. (2004). Bacterial iron homeostasis. Journal of Bacteriology, 186(5), 1438–1447. https://doi.org/10.1128/jb.186.5.1438-1447.2004
3.Rabah, A., & Hanchi, S. (2023). Experimental and modeling study of the rheological and thermophysical properties of molybdenum disulfide-based nanofluids. Journal of Molecular Liquids, 384, 123335. https://doi.org/10.1016/j.molliq.2023.123335
4. Boutte, C. C., & Crosson, S. (2009). Bacterial lifestyle shapes stringent response activation. Journal of Bacteriology, 191(9), 2904-2912. https://doi.org/10.1128/jb.01003-08
5. Mackie, J., Liu, Y. C., & DiBartolo, G. (2019). The C-terminal region of the Caulobacter crescentus CtrA protein inhibits stalk synthesis during the G1-to-S transition. mBio, 10(2), e02273-18. https://doi.org/10.1128/mbio.02273-18
6.Thanbichler, M., & Shapiro, L. (2003). MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Journal of Bacteriology, 185(4), 1432-1442. https://doi.org/10.1128/jb.185.4.1432-1442.2003
7. 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.
8. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019b) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.