Part:BBa_K2710002

His-Alpha Prefoldin with SpyCatcher

A HisTag and GSG linker was added to the N-terminus of an alpha subunit of the protein 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(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 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.

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

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.

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_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 2). 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 2: 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 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).

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.

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.

SpyCatcher/Tag Assembly

IaaH fused with SpyTag and the 6xHis aPFD-SpyCatcher were mixed at a concentration of 3 µM and 15 µM respectively in a total volume of 250 µL in PBS pH 8, and incubated at room temperature. After 0, 10, 20 and 30 minutes of incubation, a 10 µL sample was taken and boiled with 5 µL of 4x Bolt LDS sample buffer for 10 minutes at 95oC to cease SpyCatcher reactivity while preserving any covalent interactions. The samples were then examined on SDS-PAGE (Figure 5).

A higher molecular weight band, consistent with a fusion of aPFD-SpyC and IaaH-SpyT (83 kDa), emerges after 10 minutes of reaction and increases in intensity as reaction time increases. In addition, the disappearance of aPFD-SpyC band as reaction time increases suggests that a high proportion of aPFD-SpyC has reacted with the SpyTag on the enzyme.

Figure 5: aPFD-SpyC covalently attaches to IaaH-SpyT. The bands indicating successful attachment of IaaH-SpyT to aPFD-SpyC are boxed in red.

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).