Part:BBa_K2710002

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:

• 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]

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. 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).
3. 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).
4. 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).
5. 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).