Part:BBa_K5246025
CB2/CB2A HfsG Glycosyltransferase, 6xHis tag for purification
Contents
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_K5246007.
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].
HfsG, in particular, is responsible for transfer of N-acetyl-D-glucosamine to a sugar acceptor.
HfsG
HfsG gene encodes a cytoplasmic protein of 309 aa homologous to family 2 glycosyltransferases. Transferrs sugar units from UDP-GlcNAc to oligosaccharide, catalyzes the polymerization of GlcNAc. C.Crescentus HfsG mutants were completely devoid of holdfast material.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 855
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 58
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 510
- 1000COMPATIBLE WITH RFC[1000]
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.
HfsG is visible on the left side 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 |
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.
HfsG 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 |
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.
HfsG was purified and clearly seen in the elution fraction of the gel.
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.
HfsG was purified and clearly seen in the elution fraction of the gel.
References
| None |