The UNSW iGEM team successfully created RFC10 compatible BioBricks linked to our project. BioBricks were validated by sequence verification and a diagnostic gel, characterised experimentally, and physically submitted to the registry. The BioBricks we are submitting for silver are BBa_K2710000 and BBa_K2710001, which encode for the Alpha and Beta subunits of the Prefoldin protein respectively, and BBa_K2710002, His-Alpha Prefoldin with a SpyCatcher. Refer to the respective part's registry pages for more details.
Best Basic Part
This year, the UNSW iGEM team has chosen to submit the His-Alpha Prefoldin with SpyCatcher part (BBa_K2710002) for the best basic part award.
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.
Figure 1: aPFD (red) and bPFD (pink) form hexamers. Image created using PDB ID: 1FXK1
The SpyCatcher 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 (Figure 2).
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.
Figure 2: A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS5
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 purification6.
- HisTag for ease of purification
- Self assembles with beta prefoldin
- C-terminal SpyCatcher enables irreversible attachment to SpyTagged molecules
- Flexible coiled coils and (GSG)x3 linker enable the attachment of large molecules without affecting assembly
- Thermostability of the protein for use in a wide of range of temperatures
- Strong experimental characterisation by a range of techniques
- Siegert, R. et al. Structure of the Molecular Chaperone Prefoldin. Cell 103 621-632 (2000).
- Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109 e690-e697 (2012).
- Domeradzka, N. E. et al. Protein cross-linking tools for the construction of nanomaterials. Curr Opin Biotechnol. 39 61-67 (2016).
- Reddington, S. C. and Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol. 29 94-99 (2015).
- Li, L. et al. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. J. Mol. Biol. 426 309–317 (2014).
- Hochuli, E., Dobeli, H. and Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 411 177-184 (1987).