Coding

Part:BBa_K5246026

Designed by: Edgaras Zaboras   Group: iGEM24_Vilnius-Lithuania   (2024-09-22)
Revision as of 09:37, 29 September 2024 by Sarpilo (Talk | contribs) (Introduction)


CB2/CB2A HfsH Deacetylase, 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_K5246042.

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_K5246020.

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]

hfsh.png
Fig. 1. Alphafold 3 structure showing

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.

HfsH is purifyable and clearly seen in the gel elution fraction. HfsH could migrate higher than its expected size because the higher the positive charge density (more charges per molecule mass), the slower a protein will migrate. At the same time, the frictional force of the gel matrix creates a sieving effect, regulating the movement of proteins according to their size and three-dimensional shape. HfsH has a high density of positively charged amino acids causing its unexpected migration in the gel

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.

HfsH is purifyable and clearly seen in the gel elution fraction. HfsH could migrate higher than its expected size because the higher the positive charge density (more charges per molecule mass), the slower a protein will migrate. At the same time, the frictional force of the gel matrix creates a sieving effect, regulating the movement of proteins according to their size and three-dimensional shape. HfsH has a high density of positively charged amino acids causing its unexpected migration in the gel.

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. 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.

[edit]
Categories
Parameters
None