Difference between revisions of "Part:BBa K2710002"
Emilywatson (Talk | contribs) |
Emilywatson (Talk | contribs) |
||
| Line 7: | Line 7: | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | Prefoldin is a molecular chaperone protein which assists in the correct folding of nascent proteins<sup>1</sup>. Alpha prefoldin (aPFD) and beta prefoldin (bPFD) are two subclasses of prefoldin which oligomerise to form a heterohexameric structure consisting of two alpha subunits and four beta subunits<sup>1</sup>. Alpha prefoldin (15.7 kDa) and beta prefoldin (13.8 kDa) derived from the thermophilic arcahea <em>Methanobacterium thermoautotrophicum</em> self assemble into an 87 kDa hexamer. The prefoldin hexamer is assembled through interactions between beta hairpins in each subunit, whereby the beta hairpins form two 8 stranded up and down beta barrels<sup>1</sup>. Each subunit contains flexible alpha-helical coiled coils, 60 to 70 Å in length, which extend from the beta barrel platform<sup>1</sup>. The hexamer is stable at high temperatures, with a Tm ≥ 70°C<sup>1</sup>.</p> | + | Prefoldin is a molecular chaperone protein which assists in the correct folding of nascent proteins<sup>1</sup>. <b>Alpha prefoldin (aPFD)</b> and beta prefoldin (bPFD) are two subclasses of prefoldin which oligomerise to form a heterohexameric structure consisting of two alpha subunits and four beta subunits<sup>1</sup>. Alpha prefoldin (15.7 kDa) and beta prefoldin (13.8 kDa) derived from the thermophilic arcahea <em>Methanobacterium thermoautotrophicum</em> self assemble into an 87 kDa hexamer. The prefoldin hexamer is assembled through interactions between beta hairpins in each subunit, whereby the beta hairpins form two 8 stranded up and down beta barrels<sup>1</sup>. Each subunit contains flexible alpha-helical coiled coils, 60 to 70 Å in length, which extend from the beta barrel platform<sup>1</sup>. The hexamer is stable at high temperatures, with a Tm ≥ 70°C<sup>1</sup>.</p> |
<br /> | <br /> | ||
| − | <span class='h3bb'>Sequence and Features</span> | + | <p>The <b>SpyTag</b> forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins<sup>2</sup>. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from <i>Streptococcus pyogenes</i> to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide<sup>2</sup>. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp<sup>117</sup> on SpyTag and Lys<sup>31</sup> on SpyCatcher<sup>2</sup>, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements<sup>2</sup>.</p> |
| + | <p>It is particularly useful as neither component needs to be at the C or N terminus<sup>3</sup>, and the effect on the attached protein’s activity appears to be negligible10. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”<sup>4</sup>. </p> | ||
| + | <br /> | ||
| + | <p>A <b>6x HisTag</b> (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification<sup>5</sup>.</p> | ||
| + | <br /> | ||
| + | |||
| + | <h2>Characterisation</h2> | ||
| + | <span class='h3bb'><b>Sequence and Features<b/></span> | ||
<partinfo>BBa_K2710002 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2710002 SequenceAndFeatures</partinfo> | ||
| Line 22: | Line 29: | ||
<ol> | <ol> | ||
<li>Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U. & Moarefi, I. Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell 103, 621–32 (2000).</li> | <li>Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U. & Moarefi, I. Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell 103, 621–32 (2000).</li> | ||
| − | + | <li>Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697, doi:10.1073/pnas.1115485109 (2012).</li> | |
| + | <li>Domeradzka, N. E., Werten, M. W., Wolf, F. A. & de Vries, R. Protein cross-linking tools for the construction of nanomaterials. Curr Opin Biotechnol 39, 61-67, doi:10.1016/j.copbio.2016.01.003 (2016).</li> | ||
| + | <li>Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol 29, 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).</li> | ||
| + | <li>Hochuli, E., Dobeli, H. & Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411, 177-184 (1987).</li> | ||
</ol> | </ol> | ||
| − | |||
</div> | </div> | ||
Revision as of 09:13, 17 October 2018
His-Alpha Prefoldin with SpyCatcher
6xHis Alpha Prefoldin with Spy-Catcher
A HisTag and GSG linker was added to the N-terminus of the protein alpha prefoldin. A SpyCatcher and (GSG)x3 linker was added to the C-terminus.
Usage and Biology
Prefoldin is a molecular chaperone protein which assists in the correct folding of nascent proteins1. Alpha prefoldin (aPFD) and beta prefoldin (bPFD) are two subclasses of prefoldin which oligomerise to form a heterohexameric structure consisting of two alpha subunits and four beta subunits1. Alpha prefoldin (15.7 kDa) and beta prefoldin (13.8 kDa) derived from the thermophilic arcahea Methanobacterium thermoautotrophicum self assemble into an 87 kDa hexamer. The prefoldin hexamer is assembled through interactions between beta hairpins in each subunit, whereby the beta hairpins form two 8 stranded up and down beta barrels1. Each subunit contains flexible alpha-helical coiled coils, 60 to 70 Å in length, which extend from the beta barrel platform1. The hexamer is stable at high temperatures, with a Tm ≥ 70°C1.
The SpyTag forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins2. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from Streptococcus pyogenes to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide2. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp117 on SpyTag and Lys31 on SpyCatcher2, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements2.
It is particularly useful as neither component needs to be at the C or N terminus3, and the effect on the attached protein’s activity appears to be negligible10. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”4.
A 6x HisTag (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification5.
Characterisation
Sequence and Features<b/></span>
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U. & Moarefi, I. Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell 103, 621–32 (2000).
- Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697, doi:10.1073/pnas.1115485109 (2012).
- Domeradzka, N. E., Werten, M. W., Wolf, F. A. & de Vries, R. Protein cross-linking tools for the construction of nanomaterials. Curr Opin Biotechnol 39, 61-67, doi:10.1016/j.copbio.2016.01.003 (2016).
- Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol 29, 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).
- Hochuli, E., Dobeli, H. & Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411, 177-184 (1987).