SpyCatcher

The SpyTag/SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair.[1] DNA sequence encoding either SpyTag or SpyCatcher can be recombinantly introduced into the DNA sequence encoding a protein of interest, forming a fusion protein. These fusion proteins can be covalently linked when mixed in a reaction through the SpyTag/SpyCatcher system.

Using the Tag/Catcher pair, bioconjugation can be achieved between two recombinant proteins that would otherwise be restrictive or impossible with traditional direct genetic fusion between the two proteins. For example, issues regarding protein folding, suboptimal expression host, and specialized post-translational modifications can be alleviated by separating the production of the proteins with the modularity of the Tag/Catcher system.[2]

Development and reaction mechanism

SpyTag and SpyCatcher were formed from the splitting and engineering of the CnaB2 domain of the FbaB protein from Streptococcus pyogenes, which naturally forms an intramolecular isopeptide bond to assist colonization of the host cell.[3][4] With the formation of the isopeptide bond, the CnaB2 domain becomes more tolerant to conformational, thermal and pH changes.

Building upon this, SpyTag was obtained from CnaB2 by extracting the C-terminal beta strand containing the reactive aspartic acid at D556 and leaving the rest of the beta strands containing the reactive lysine K470 and the catalytic glutamic acid at E516 to become SpyCatcher, after further engineering to remove some hydrophobic surface residues. The resulting SpyTag/SpyCatcher can react to form the isopeptide bond with a second-order rate constant of 1.4 ± 0.4 × 103 M−1 s−1. It is postulated[5] that the reaction mechanism proceeds by a nucleophilic attack of D556 from K470, mediated by E516. By reconstituting SpyTag:SpyCatcher, the resulting conjugated complex acquires the stability of the parent CnaB2 domain.

A second generation SpyTag/SpyCatcher called SpyTag002/SpyCatcher002 was then created through phage display that enables the peptide-protein pair to react up to 12 times faster than the original pair, at a rate constant of 2.0 ± 0.2 × 104 M−1 s−1.[6] The second generation SpyCatcher002 also has abolished self-reactivity that is present with SpyCatcher.

A third generation SpyTag/SpyCatcher called SpyTag003/SpyCatcher003 has now also been created through rational design. This reacts up to 400 fold faster than the original pair with a rate constant of 5.5 ± 0.6 × 105 M−1 s−1.[7] This version is back reactive with the two previous generations of SpyTag/SpyCatcher reagents.

SpyTag/SpyCatcher react with high specificity even when in the presence of bacterial and mammalian cell environments.[1][6][7]

Applications

Because of the specificity, irreversible covalent linkage and ease of use, the SpyTag/SpyCatcher conjugation system has seen many different applications.[5][8][9]

Vaccine production

By fusing either SpyTag or SpyCatcher to self-assembling molecules such as virus-like particles, antigens fused to the other pair can be decorated onto the molecule via the isopeptide bond formed.[10][11][12][13] This enables fast production of vaccines as the central self-assembling molecule can be stocked up beforehand, whilst the antigen can be easily produced under optimal conditions to achieve proper protein folding.

Enzyme cyclization

Cyclization of enzymes by fusing the N- and C-termini of the protein helps elevate the stability of the enzyme against heat. By having SpyTag and SpyCatcher together in the enzyme at the termini, the enzyme will undergo spontaneous cyclization by forming the isopeptide bond. Cyclized beta-lactamase, phytase, firefly luciferase, and xylanase (to name a few) have shown retained enzyme activity even after being subjected to heat at 100 °C.[14][15]

Protein hydrogels

Hydrogels have had a wide-range of applications in biomedical sciences. One commonly used type of hydrogel starting material are the elastin-like polypeptides. SpyTag/SpyCatcher chemistry has been used to produce tailored molecular networks (“networks of spies”) within these hydrogels that enable the encapsulation of living mammalian cells such as fibroblasts.[16] Subsequent modifications have enabled photo-responsive hydrogel formation,[17] user-defined control over cell-material interactions,[18] combined hyaluronan-elastin-like polypeptides,[19] as well creating protein scaffolds for enzyme flow biocatalysis.[20]

Expanded Tag/Catcher pairs

Before the development of SpyTag/SpyCatcher, the pair Isopeptag/Pilin-C was created from protein Spy0128 of Streptococcus pyogenes.[21] Following SpyTag/SpyCatcher, the fully orthogonal pair SnoopTag/SnoopCatcher was developed from the RrgA protein of Streptococcus pneumoniae that has no cross-reactivity with SpyTag/SpyCatcher.[22] Note that SnoopTag/SnoopCatcher forms an isopeptide bond between a Lys-Asn instead of Lys-Asp found in SpyTag/SpyCatcher. The same domain from RrgA has now been split in a different way to that used to create SnoopTag/SnoopCatcher, with the new pair called DogTag/DogCatcher. Unlike SpyTag and SnoopTag which have extended structures, the region of RrgA used to create DogTag forms a β-hairpin and so predisposed for successful insertion into protein loops. This ability has been successfully exploited to fluorescently label an internal loop of the mammalian TRPC5 membrane channel protein which cannot be modified at the protein termini, without impacting on the channel properties of TRPC5.[23] DogTag has been successful coupled to DogCatcher when inserted into soluble proteins (superfolder GFP, HaloTag, and Gre2p).[23]

The pair SdyTag/SdyCatcher was also developed in the same year from Streptococcus dysgalactiae fibronectin-binding protein CnaB domain, but since the protein has sequence similarity to the parent protein where SpyTag/SpyCatcher is derived from, this Tag/Catcher pair has cross-reactivity with the latter pair.[24]

Rather than finding homologous proteins from different species, a new Tag/Catcher pair was developed from SpyTag/SpyCatcher with minimal mutations. SpyTag I3W (AW) reacts with SpyCatcher F77V, F94A (BVA) but minimally with SpyCatcher, whereas SpyCatcher F77V, F94A can react with both SpyTag I3W and SpyTag.[25] However, the cross-reactivity of SpyCatcher F77V, F94A with both SpyTag versions may limit its utility as a new Tag/Catcher pair.

A different chemistry can be exploited for protein ligation: the discovery of an intramolecular ester bond formation in Clostridium perfringens cell-surface adhesin protein Cpe0147 led to the development of another Tag/Catcher pair with Cpe0147565–587 as the Tag and Cpe0147439–563 as the Catcher.[26] The ester bond formed between Thr-Gln is irreversible, however by mutating the Thr to Ser, the Ser-Gln ester bond is reversible with a change of pH.

Spy&Go affinity purification

Mutation of the catalytic glutamic acid residue (E77) in SpyCatcher to alanine stops isopeptide bond formation but does not prevent the initial non-covalent SpyTag/SpyCatcher association. This non-covalent SpyTag/SpyCatcher interaction has been utilized in the affinity purification of SpyTag-fused recombinant proteins.[27] In this purification strategy, termed Spy&Go, resin-immobilized SpyCatcher is used to harvest SpyTag-fused proteins from cell culture supernatants or cell lysates. Non-specifically bound proteins are removed by washing the resin with a neutral buffer and the target protein eluted at neutral pH using high imidazole concentration.

The Spy&Go affinity resin is based on SpyCatcher2.1 E77A S49C variant termed SpyDock.[27] SpyDock can be expressed in E. coli as soluble protein, purified using Ni-NTA and anion-exchange resins and immobilized to iodoacetyl-activated agarose through the unpaired cysteine introduced by the S49C substitution. In neutral buffers with physiological salt concentration SpyDock binds to SpyTag- and SpyTag002-fused proteins with affinity in the high nanomolar range (Kd = 750 ± 50 nM for SpyTag, Kd = 73 ± 13 nM for SpyTag002).[27] Affinity to SpyTag003 has not been reported, but requires harsher conditions to ensure full dissociation suggesting it binds tighter.[7] SpyDock-bound proteins are eluted by incubating the resin with 2.5 M imidazole in neutral buffer.[27] The SpyDock resin can be regenerated several times using consecutive washes with 4 M imidazole, 6 M guanidinium hydrochloride and 0.1 M NaOH.[27]

Spy&Go purification of proteins with either N-terminal, internal or C-terminal SpyTag have all been reported.[7][27] SpyDock resin is compatible with all SpyTag generations (SpyTag,[1] SpyTag002,[6] SpyTag003[7]) and it does not interfere with the later covalent conjugation of the purified proteins with SpyCatcher.[27]

See also
  • Protein tag
  • Bioconjugation
  • Biomolecular engineering
  • Isopeptide bond

References
  1. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M (March 2012). "Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin". Proceedings of the National Academy of Sciences of the United States of America. 109 (12): E690-7. doi:10.1073/pnas.1115485109. PMC 3311370. PMID 22366317.
  2. Brune KD, Howarth M (26 June 2018). "New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue". Frontiers in Immunology. 9: 1432. doi:10.3389/fimmu.2018.01432. PMC 6028521. PMID 29997617.
  3. Kang HJ, Baker EN (April 2011). "Intramolecular isopeptide bonds: protein crosslinks built for stress?". Trends in Biochemical Sciences. 36 (4): 229–37. doi:10.1016/j.tibs.2010.09.007. PMID 21055949.
  4. Hagan RM, Björnsson R, McMahon SA, Schomburg B, Braithwaite V, Bühl M, et al. (November 2010). "NMR spectroscopic and theoretical analysis of a spontaneously formed Lys-Asp isopeptide bond". Angewandte Chemie. 49 (45): 8421–5. doi:10.1002/anie.201004340. PMC 3315829. PMID 20878961.
  5. Reddington SC, Howarth M (December 2015). "Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher". Current Opinion in Chemical Biology. 29: 94–9. doi:10.1016/j.cbpa.2015.10.002. PMID 26517567.
  6. Keeble AH, Banerjee A, Ferla MP, Reddington SC, Anuar IN, Howarth M (December 2017). "Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics". Angewandte Chemie. 56 (52): 16521–16525. doi:10.1002/anie.201707623. PMC 5814910. PMID 29024296.
  7. Keeble AH, Turkki P, Stokes S, Khairil Anuar IN, Rahikainen R, Hytönen VP, Howarth M (December 2019). "Approaching infinite affinity through engineering of peptide-protein interaction". Proceedings of the National Academy of Sciences of the United States of America. 116 (52): 26523–26533. doi:10.1073/pnas.1909653116. PMC 6936558. PMID 31822621.
  8. Banerjee A, Howarth M (June 2018). "Nanoteamwork: covalent protein assembly beyond duets towards protein ensembles and orchestras". Current Opinion in Biotechnology. 51: 16–23. doi:10.1016/j.copbio.2017.10.006. PMID 29172131.
  9. Keeble AH, Howarth M (2019). "Insider information on successful covalent protein coupling with help from SpyBank". Methods in Enzymology. 617: 443–461. doi:10.1016/bs.mie.2018.12.010. ISBN 9780128170748. PMID 30784412. S2CID 73473893.
  10. Brune KD, Buldun CM, Li Y, Taylor IJ, Brod F, Biswas S, Howarth M (May 2017). "Dual Plug-and-Display Synthetic Assembly Using Orthogonal Reactive Proteins for Twin Antigen Immunization". Bioconjugate Chemistry. 28 (5): 1544–1551. doi:10.1021/acs.bioconjchem.7b00174. PMID 28437083.
  11. Thrane S, Janitzek CM, Matondo S, Resende M, Gustavsson T, de Jongh WA, et al. (April 2016). "Bacterial superglue enables easy development of efficient virus-like particle based vaccines". Journal of Nanobiotechnology. 14 (1): 30. doi:10.1186/s12951-016-0181-1. PMC 4847360. PMID 27117585.
  12. Brune KD, Leneghan DB, Brian IJ, Ishizuka AS, Bachmann MF, Draper SJ, et al. (January 2016). "Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization". Scientific Reports. 6 (1): 19234. Bibcode:2016NatSR...619234B. doi:10.1038/srep19234. PMC 4725971. PMID 26781591.
  13. Tan, Tiong Kit; Rijal, Pramila; Rahikainen, Rolle; Keeble, Anthony H.; Schimanski, Lisa; Hussain, Saira; et al. (22 January 2021). "A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses". Nature Communications. 12 (1): 542. Bibcode:2021NatCo..12..542T. doi:10.1038/s41467-020-20654-7. ISSN 2041-1723. PMC 7822889. PMID 33483491.
  14. Schoene C, Bennett SP, Howarth M (16 June 2016). "SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience". Methods in Enzymology. 580: 149–67. doi:10.1016/bs.mie.2016.05.004. ISBN 9780128053805. PMID 27586332.
  15. Gilbert C, Howarth M, Harwood CR, Ellis T (June 2017). "Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis". ACS Synthetic Biology. 6 (6): 957–967. doi:10.1021/acssynbio.6b00292. PMID 28230977.
  16. Sun F, Zhang WB, Mahdavi A, Arnold FH, Tirrell DA (August 2014). "Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry". Proceedings of the National Academy of Sciences of the United States of America. 111 (31): 11269–74. Bibcode:2014PNAS..11111269S. doi:10.1073/pnas.1401291111. PMC 4128157. PMID 25049400.
  17. Wang R, Yang Z, Luo J, Hsing IM, Sun F (June 2017). "12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release". Proceedings of the National Academy of Sciences of the United States of America. 114 (23): 5912–5917. doi:10.1073/pnas.1621350114. PMC 5468657. PMID 28533376.
  18. Hammer JA, Ruta A, Therien AM, West JL (July 2019). "Cell-Compatible, Site-Specific Covalent Modification of Hydrogel Scaffolds Enables User-Defined Control over Cell-Material Interactions". Biomacromolecules. 20 (7): 2486–2493. doi:10.1021/acs.biomac.9b00183. PMID 31121097. S2CID 163167760.
  19. Wieduwild R, Howarth M (October 2018). "Assembling and decorating hyaluronan hydrogels with twin protein superglues to mimic cell-cell interactions". Biomaterials. 180: 253–264. doi:10.1016/j.biomaterials.2018.07.020. PMID 30053659. S2CID 51726676.
  20. Peschke T, Bitterwolf P, Gallus S, Hu Y, Oelschlaeger C, Willenbacher N, et al. (December 2018). "Self-Assembling All-Enzyme Hydrogels for Flow Biocatalysis". Angewandte Chemie. 57 (52): 17028–17032. doi:10.1002/anie.201810331. PMID 30380178. S2CID 53150078.
  21. Zakeri B, Howarth M (April 2010). "Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting". Journal of the American Chemical Society. 132 (13): 4526–7. CiteSeerX 10.1.1.706.4839. doi:10.1021/ja910795a. PMID 20235501.
  22. Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M (February 2016). "Programmable polyproteams built using twin peptide superglues". Proceedings of the National Academy of Sciences of the United States of America. 113 (5): 1202–7. Bibcode:2016PNAS..113.1202V. doi:10.1073/pnas.1519214113. PMC 4747704. PMID 26787909.
  23. Keeble, Anthony H.; Yadav, Vikash K.; Ferla, Matteo P.; Bauer, Claudia C.; Chuntharpursat-Bon, Eulashini; Huang, Jin; Bon, Robin S.; Howarth, Mark (July 2021). "DogCatcher allows loop-friendly protein-protein ligation". Cell Chemical Biology. doi:10.1016/j.chembiol.2021.07.005. ISSN 2451-9456. PMID 34324879.
  24. Tan LL, Hoon SS, Wong FT (26 October 2016). "Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly". PLOS ONE. 11 (10): e0165074. Bibcode:2016PLoSO..1165074T. doi:10.1371/journal.pone.0165074. PMC 5082641. PMID 27783674.
  25. Liu Y, Liu D, Yang W, Wu XL, Lai L, Zhang WB (September 2017). "Tuning SpyTag-SpyCatcher mutant pairs toward orthogonal reactivity encryption". Chemical Science. 8 (9): 6577–6582. doi:10.1039/C7SC02686B. PMC 5627348. PMID 28989685.
  26. Young PG, Yosaatmadja Y, Harris PW, Leung IK, Baker EN, Squire CJ (January 2017). "Harnessing ester bond chemistry for protein ligation". Chemical Communications. 53 (9): 1502–1505. doi:10.1039/C6CC09899A. PMID 28084475.
  27. Khairil Anuar IN, Banerjee A, Keeble AH, Carella A, Nikov GI, Howarth M (April 2019). "Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox". Nature Communications. 10 (1): 1734. Bibcode:2019NatCo..10.1734K. doi:10.1038/s41467-019-09678-w. PMC 6465384. PMID 30988307.


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