Difference between revisions of "Part:BBa K2710000"
Emilywatson (Talk | contribs) |
Emilywatson (Talk | contribs) |
||
| Line 7: | Line 7: | ||
<h3>Usage and Biology</h3> | <h3>Usage and Biology</h3> | ||
| − | <p>Prefoldin is a hetero-hexameric chaperone protein consisting of two α and four β subunits1, which was isolated from the thermophilic bacteria <i>Methanobacterium thermoautotrophicum</i><sup>2</sup>. As a result, prefoldin is stable at high temperatures, with a Tm ≥ 70°C<sup>2</sup>. This 85kDa molecular chaperone protein is self-assembling, and consists of six α-helix coiled-coils (with each subunit as a coiled coil) protruding from a double β-barrel<sup>3</sup>. Each α subunit forms two β hairpins, and the four β hairpins from the two α subunits forms the two 8-stranded up and down β barrels<sup>2</sup>. Additionally, each β subunit forms one β hairpin. The subunits are anchored to the barrel via their proximal ends, and are fully solvated<sup>2</sup>. | + | <p><b>Prefoldin</b> is a hetero-hexameric chaperone protein consisting of two α and four β subunits1, which was isolated from the thermophilic bacteria <i>Methanobacterium thermoautotrophicum</i><sup>2</sup>. As a result, prefoldin is stable at high temperatures, with a Tm ≥ 70°C<sup>2</sup>. This 85kDa molecular chaperone protein is self-assembling, and consists of six α-helix coiled-coils (with each subunit as a coiled coil) protruding from a double β-barrel<sup>3</sup>. Each α subunit forms two β hairpins, and the four β hairpins from the two α subunits forms the two 8-stranded up and down β barrels<sup>2</sup>. Additionally, each β subunit forms one β hairpin. The subunits are anchored to the barrel via their proximal ends, and are fully solvated<sup>2</sup>. |
The α subunits are 85 Angstrom in height, and the β subunits are 70 Angstrom in height, with an overall distance of 50 Angstrom across the double β-barrel<sup>2</sup>. Sigert’s research also suggests that the coiled coils are flexible, although the overall structure is highly conserved<sup>2</sup>. Prefoldin is a molecular chaperone which, in archaea, carries out ‘de novo protein folding’, and in eukaryotes assists in the biogenesis of cytoskeletal proteins, thanks to its coiled coil ‘tentacles’<sup>1</sup>. We, however, are using it as the backbone to our modular, thermostable enzyme scaffold.</p> | The α subunits are 85 Angstrom in height, and the β subunits are 70 Angstrom in height, with an overall distance of 50 Angstrom across the double β-barrel<sup>2</sup>. Sigert’s research also suggests that the coiled coils are flexible, although the overall structure is highly conserved<sup>2</sup>. Prefoldin is a molecular chaperone which, in archaea, carries out ‘de novo protein folding’, and in eukaryotes assists in the biogenesis of cytoskeletal proteins, thanks to its coiled coil ‘tentacles’<sup>1</sup>. We, however, are using it as the backbone to our modular, thermostable enzyme scaffold.</p> | ||
| + | |||
| + | |||
| + | <p><b>Hexahistidine tags (‘His-tags’)</b> are small metal-chelating tags of five or six consecutive histidine residues which can be expressed onto other proteins by fusing them to their N or C terminus4. They have “minimal effect on [protein] tertiary structure and biological activity” depending on location and are “easy and specific to remove”5. Histidine was chosen specifically because it has the “strongest interactions with metal ion matrices” but these interactions can be disrupted using pH or imidazole5. | ||
| + | Often this is done in order to make protein purification of recombinant proteins easier, because His-tagged proteins can be selected from many others using a ‘His-trap’ (metal affinity chromatography)6. This tag works because the hexahistidine binds to the metals Cu2+, Co2+, Zn2+ and Ni2+, and as a result, the selected proteins expressing the His-tag can be isolated because they alone are supposed to remain bound to the metal of the column or surface4. The selection of His rich proteins produced by certain expression strains (e.g. Escherichia coli) can prove more difficult if the concentration produced of the desired protein is very small, and the strain has many His-rich proteins7.</p> | ||
<!-- --> | <!-- --> | ||
Revision as of 04:33, 16 October 2018
His-Alpha Prefoldin
alpha-Prefoldin with His-Tag
Usage and Biology
Prefoldin is a hetero-hexameric chaperone protein consisting of two α and four β subunits1, which was isolated from the thermophilic bacteria Methanobacterium thermoautotrophicum2. As a result, prefoldin is stable at high temperatures, with a Tm ≥ 70°C2. This 85kDa molecular chaperone protein is self-assembling, and consists of six α-helix coiled-coils (with each subunit as a coiled coil) protruding from a double β-barrel3. Each α subunit forms two β hairpins, and the four β hairpins from the two α subunits forms the two 8-stranded up and down β barrels2. Additionally, each β subunit forms one β hairpin. The subunits are anchored to the barrel via their proximal ends, and are fully solvated2. The α subunits are 85 Angstrom in height, and the β subunits are 70 Angstrom in height, with an overall distance of 50 Angstrom across the double β-barrel2. Sigert’s research also suggests that the coiled coils are flexible, although the overall structure is highly conserved2. Prefoldin is a molecular chaperone which, in archaea, carries out ‘de novo protein folding’, and in eukaryotes assists in the biogenesis of cytoskeletal proteins, thanks to its coiled coil ‘tentacles’1. We, however, are using it as the backbone to our modular, thermostable enzyme scaffold.
Hexahistidine tags (‘His-tags’) are small metal-chelating tags of five or six consecutive histidine residues which can be expressed onto other proteins by fusing them to their N or C terminus4. They have “minimal effect on [protein] tertiary structure and biological activity” depending on location and are “easy and specific to remove”5. Histidine was chosen specifically because it has the “strongest interactions with metal ion matrices” but these interactions can be disrupted using pH or imidazole5. Often this is done in order to make protein purification of recombinant proteins easier, because His-tagged proteins can be selected from many others using a ‘His-trap’ (metal affinity chromatography)6. This tag works because the hexahistidine binds to the metals Cu2+, Co2+, Zn2+ and Ni2+, and as a result, the selected proteins expressing the His-tag can be isolated because they alone are supposed to remain bound to the metal of the column or surface4. The selection of His rich proteins produced by certain expression strains (e.g. Escherichia coli) can prove more difficult if the concentration produced of the desired protein is very small, and the strain has many His-rich proteins7.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]