Difference between revisions of "Part:BBa K2710002"
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| − | < | + | <h3>Characterisation</h3> |
| − | <span class='h3bb'><b>Sequence and Features< | + | <p><b>Protein Purification</b></p> |
| + | <p>BBa_K2710002 (encoding 6xHis aPFD-SpyCatcher) 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 1). The 6xHis aPFD-SpyCatcher was successfully purified as reflected by the clear bands seen at the expected molecular weight range (30 kDa) in the elution lanes.</p> | ||
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| + | https://static.igem.org/mediawiki/2018/e/e9/T--UNSW_Australia--aPFD-SpyC.jpeg | ||
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| + | <p class="figure-legend fig1-leg"><b>Figure 1:</b> SDS-PAGE analysis of IMAC purification of 6xHis Alpha Prefoldin with Spy-Catcher (BBa_K2710002). 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 aPFD-SpyCatcher (MW: 30 kDa).</p> | ||
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| + | <p><b>SEC</b></p> | ||
| + | <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> | ||
| + | <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 4oC. 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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| + | <span class='h3bb'><b>Sequence and Features</b></span> | ||
<partinfo>BBa_K2710002 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2710002 SequenceAndFeatures</partinfo> | ||
Revision as of 09:42, 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
Protein Purification
BBa_K2710002 (encoding 6xHis aPFD-SpyCatcher) 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 1). The 6xHis aPFD-SpyCatcher was successfully purified as reflected by the clear bands seen at the expected molecular weight range (30 kDa) in the elution lanes.
Figure 1: SDS-PAGE analysis of IMAC purification of 6xHis Alpha Prefoldin with Spy-Catcher (BBa_K2710002). 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 aPFD-SpyCatcher (MW: 30 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 4oC. 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).
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]
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).