Difference between revisions of "Part:BBa K2710001"
 
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<partinfo>BBa_K2710001 short</partinfo>
 
<partinfo>BBa_K2710001 short</partinfo>
  
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6xHis Beta Prefoldin
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<p> A HisTag and GSG linker was added to the N-terminus of the beta prefoldin subunit, prefoldin being a protein. </p>
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<h3>Usage and Biology</h3>
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<p>Prefoldin is a molecular chaperone protein which assists in the correct folding of nascent proteins<sup>1</sup>. Alpha prefoldin (aPFD) and <b>beta prefoldin (bPFD)</b> are two subclasses of prefoldin which oligomerise to form a heterohexameric structure consisting of two alpha subunits and four beta subunits<sup>1</sup>(Figure 1). 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, eight 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&deg;C<sup>1</sup>. This makes it suitable for use in an scaffold system.</p>
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<br />
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https://static.igem.org/mediawiki/2018/e/e8/T--UNSW_Australia--bec-design-pfdhex.png
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<p class="figure-legend fig1-leg"><b>Figure 1:</b> aPFD (red) and bPFD (pink) form hexamers. Image created using PDB ID: 1FXK<sup>2</sup></p>
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<br />
  
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<p>A <b>6xHisTag</b> (six consecutive histidine residues, also known as a hexahistidine tag) was added to bPFD to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification<sup>2</sup>.</p>
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<br />
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<span class='h3bb'><b>Sequence and Features</b></span>
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<partinfo>BBa_K2710002 SequenceAndFeatures</partinfo>
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<h3>Usage and Biology</h3>
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<h3>Characterisation</h3>
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<p><b>Protein Purification</b></p>
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<p>BBa_K2710001 (encoding 6xHis bPFD) was subcloned into the multiple cloning site of pET19b for expression in E. coli T7 Express (NEB) and purified by Immobilised Metal Affinity Chromatography (IMAC). The purification was analysed by SDS-PAGE (Figure 2). The 6xHis aPFD was successfully purified as reflected by the clear bands seen at the expected molecular weight range (15 kDa) in the elution lanes.</p>
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<br />
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https://static.igem.org/mediawiki/2018/thumb/8/8e/T--UNSW_Australia--aPFD-bPFD-purification.jpeg/739px-T--UNSW_Australia--aPFD-bPFD-purification.jpeg
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<p class="figure-legend fig2-leg"><b>Figure 2:</b> SDS-PAGE analysis of IMAC purification of 6xHis aPFD (left) and 6xHis bPFD (right). SeeBlue Plus 2 Pre-stained Protein Standard (Invitrogen) was used as the molecular weight standard. Lanes are labelled as flow through (FT1 and FT2), wash (W) and elutions (E1, E2, E3).  Successful purification of 6xHis bPFD confirmed (MW: 15 kDa).</p>
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<br />
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<p><b>SEC</b></p>
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<p>Size Exclusion Chromatography (SEC) is an appropriate method for analysing the assembly of prefoldin hexamers as it retains the native structure of assemblies and can distinguish between molecules of varying size. SEC was used to demonstrate the formation of aPFD and bPFD hexamers, and to investigate of the monodispersity of the sample.</p>
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<p>IMAC purified alpha prefoldin and beta prefoldin were mixed in a 1:2 molar ratio to a total volume of 1 mL at concentrations of 1 mg/mL in PBS pH 8 and incubated overnight at 4&deg;C. Size Exclusion Chromatography was kindly performed by Ms Hélène Lebhar. Alpha prefoldin, beta prefoldin and the mixture were loaded onto a Superdex S200 Increase 10/300 GL column using an AKTA start, and separated by SEC. The chromatograms of the three runs were then overlayed for analysis, and compared to the molecular weight standards thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa) and myoglobulin (17 kDa).</p>
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<br />
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https://static.igem.org/mediawiki/2018/thumb/5/55/T--UNSW_Australia--assembly1.jpeg/800px-T--UNSW_Australia--assembly1.jpeg
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<p class="figure-legend fig3-leg"><b>Figure 3:</b>Overlayed SEC chromatograms of aPFD (pink), bPFD (brown) and a 1:2 molar ratio mixture of aPFD and bFPD (orange).</p>
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<br />
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<p>SEC chromatograms of aPFD, bPFD and a 1:2 molar mixture of aPFD and bPFD were overlayed, revealing the formation of larger molecular weight structures in the mixture of aPFD and bFPD. The peak was not of a Gaussian distribution, suggesting that the larger structures were not monodisperse, and that several oligomers exist. Four peaks were identified as potential oligomeric structures, with the largest peak eluting at 13.3 mL.</p>
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<br />
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https://static.igem.org/mediawiki/2018/0/04/T--UNSW_Australia--sec_calibration_cutve.png
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<p class="figure-legend fig4-leg"><b>Figure 4:</b> SEC calibration curve obtained using molecular weight standards thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa) and myoglobulin (17 kDa).</p>
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<br />
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<p>The predicted molecular weight of the largest alpha beta peak is consistent with the size of a hexamer, but the peak does not appear to be monodispersed (Table 1). It appears that largest structures are present.</p>
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<br />
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<p class="figure-legend"><b>Table 1:</b> Predicted molecular weights of peaks from SEC using the SEC calibration curve.</p>
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https://static.igem.org/mediawiki/parts/thumb/d/dd/T--UNSW_Australia--biobricktable1.png/800px-T--UNSW_Australia--biobricktable1.png
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<p>Prefoldin is a hetero-hexameric 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>.
 
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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>
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K2710001 SequenceAndFeatures</partinfo>
 
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2710001 parameters</partinfo>
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<partinfo>BBa_K2710002 parameters</partinfo>
 
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<div id="references">
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<h2>References</h2>
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<ol>
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<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>
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<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>
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</ol>
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</div>

Latest revision as of 16:28, 17 October 2018


His-Beta Prefoldin

A HisTag and GSG linker was added to the N-terminus of the beta prefoldin subunit, prefoldin being a protein.

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(Figure 1). 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, eight 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. This makes it suitable for use in an scaffold system.


T--UNSW_Australia--bec-design-pfdhex.png

Figure 1: aPFD (red) and bPFD (pink) form hexamers. Image created using PDB ID: 1FXK2


A 6xHisTag (six consecutive histidine residues, also known as a hexahistidine tag) was added to bPFD to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification2.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterisation

Protein Purification

BBa_K2710001 (encoding 6xHis bPFD) was subcloned into the multiple cloning site of pET19b for expression in E. coli T7 Express (NEB) and purified by Immobilised Metal Affinity Chromatography (IMAC). The purification was analysed by SDS-PAGE (Figure 2). The 6xHis aPFD was successfully purified as reflected by the clear bands seen at the expected molecular weight range (15 kDa) in the elution lanes.


739px-T--UNSW_Australia--aPFD-bPFD-purification.jpeg

Figure 2: SDS-PAGE analysis of IMAC purification of 6xHis aPFD (left) and 6xHis bPFD (right). SeeBlue Plus 2 Pre-stained Protein Standard (Invitrogen) was used as the molecular weight standard. Lanes are labelled as flow through (FT1 and FT2), wash (W) and elutions (E1, E2, E3). Successful purification of 6xHis bPFD confirmed (MW: 15 kDa).


SEC

Size Exclusion Chromatography (SEC) is an appropriate method for analysing the assembly of prefoldin hexamers as it retains the native structure of assemblies and can distinguish between molecules of varying size. SEC was used to demonstrate the formation of aPFD and bPFD hexamers, and to investigate of the monodispersity of the sample.

IMAC purified alpha prefoldin and beta prefoldin were mixed in a 1:2 molar ratio to a total volume of 1 mL at concentrations of 1 mg/mL in PBS pH 8 and incubated overnight at 4°C. Size Exclusion Chromatography was kindly performed by Ms Hélène Lebhar. Alpha prefoldin, beta prefoldin and the mixture were loaded onto a Superdex S200 Increase 10/300 GL column using an AKTA start, and separated by SEC. The chromatograms of the three runs were then overlayed for analysis, and compared to the molecular weight standards thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa) and myoglobulin (17 kDa).


800px-T--UNSW_Australia--assembly1.jpeg

Figure 3:Overlayed SEC chromatograms of aPFD (pink), bPFD (brown) and a 1:2 molar ratio mixture of aPFD and bFPD (orange).


SEC chromatograms of aPFD, bPFD and a 1:2 molar mixture of aPFD and bPFD were overlayed, revealing the formation of larger molecular weight structures in the mixture of aPFD and bFPD. The peak was not of a Gaussian distribution, suggesting that the larger structures were not monodisperse, and that several oligomers exist. Four peaks were identified as potential oligomeric structures, with the largest peak eluting at 13.3 mL.


T--UNSW_Australia--sec_calibration_cutve.png

Figure 4: SEC calibration curve obtained using molecular weight standards thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa) and myoglobulin (17 kDa).


The predicted molecular weight of the largest alpha beta peak is consistent with the size of a hexamer, but the peak does not appear to be monodispersed (Table 1). It appears that largest structures are present.


Table 1: Predicted molecular weights of peaks from SEC using the SEC calibration curve.

800px-T--UNSW_Australia--biobricktable1.png


References

  1. 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).
  2. 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).