Difference between revisions of "Part:BBa K5246026"
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<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> | ||
| − | <p><b>HfsH</b> is purifyable and clearly seen in the gel elution fraction | + | <p><b>HfsH</b> 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</p> |
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<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> | ||
| − | <p><b>HfsH</b> is purifyable and clearly seen in the gel elution fraction.</p> | + | <p><b>HfsH</b> 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.</p> |
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Revision as of 09:29, 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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 10
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 10
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 10
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 10
- 25INCOMPATIBLE 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 - 1000COMPATIBLE 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.
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
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.
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.