SpyTag/SpyCatcher Protein Coupling Reagents

SpyTag/SpyCatcher Protein Coupling Reagents

The SpyTag/SpyCatcher system is a convenient protein coupling tool for irreversible peptide-protein ligation. It is ideal for binding, labeling, immobilization and creating new kinds of protein architectures.

Highlights:

  • SpyTag forms a spontaneous amide bond upon binding its genetically encoded partner SpyCatcher
  • Highly compatible - SpyTag reacts with SpyCatcher under a wide range of conditions
  • Extremely stable reaction product - SpyCatcher/SpyTag complex is stable to boiling in SDS
  • SpyCatcher (S49C) version contains a unique cysteine residue for precise labeling with dye or precise attachment to surfaces or beads

Peptide interaction with proteins is usually weak. SpyTag is a genetically encoded peptide (SpyTag003 sequence RGVPHIVMVDAYKRYK) that forms a spontaneous amide bond upon binding its genetically encoded partner SpyCatcher (with the latest version SpyCatcher003 available here). SpyTag003 reacts with SpyCatcher003 under a wide range of conditions and the after reaction the product is stable to boiling in SDS. SpyCatcher003 (S49C) has a unique cysteine for precise labeling with dye or precise attachment to surfaces or beads.

From the laboratory of Mark Howarth, PhD, University of Oxford.

Catalog Number Product DataSheet Size AVAILABILITY Price Qty
EOX003
SpyCatcher003 Protein
0.5mg In stock
Regular Price:$105.00
On Sale:
EOX004
SpyCatcher003 (S49C) Protein
0.5mg Unavailable
Regular Price:$105.00
On Sale:
EOX005
SpyTag003-MBP Protein
0.5mg In stock
Regular Price:$175.00
On Sale:
Specifications

Product Type: Protein
Name: SpyTag/SpyCatcher Protein Coupling Reagents
Accession ID: JQ478411.1 (SpyCatcher)
Source: Recombinant expression in E. coli
Molecular Weight: 15,597.7 Da (SpyCatcher003) 15,613.7 Da (SpyCatcher003, S49C); 44,867.78 Da (SpyTag003-MBP)
Amino Acid Sequence: SpyCatcher003: SYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS
SpyCatcher003 (S49C): SYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDCSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS
SpyTag003: GSSHHHHHHSSGLVPRGSRGVPHIVMVDAYKRYKGSGESGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSS
Fusion Tag(s): N-terminal 6xHis tag for each
Purity: >95%. Purified from Ni-NTA affinity chromatography and then by size exclusion chromatography.
Buffer: Phosphate buffered saline (PBS) pH 7.4
Concentration: 2.32mg/mL (SpyCatcher003), 1.8mg/mL (SpyCatcher003, S49C), 2.47mg/mL (SpyTag003-MBP)
Amount: 0.5mg
Storage: -80C, avoid multiple freeze-thaw cycles
Shipped: Dry ice

Data
SpyCatcher/SpyTag
Validation of SpyCatcher003 and SpyTag003 samples 10 uM SpyTag003-MBP, SpyCatcher003, and SpyCatcher003 (S49C) alone and reacted with 2-fold excess of the other component and ran on 16% SDS-PAGE. Note: In the absence of reductant a small amount of SpyCatcher003 is dimeric due to disulfide bond.
Provider
From the laboratory of Mark Howarth, PhD, University of Oxford.
Comments

SpyCatcher003 reacts with SpyTag in the pH range 5-8 and is relatively insensitive to the buffer composition, as long as SDS or denaturants like urea or guanidinium are not used.

SpyCatcher003: SDS-PAGE shows one higher band from gluconylation, which does not affect reactivity with SpyTag.

  • Theoretical pI: 4.52
  • Extinction coefficient at 280 nm (predicted by ProtParam): 17,420 M-1 cm-1

SpyCatcher003 (S49C): SDS-PAGE shows a 2nd band at slightly higher MW related to glucosylation, but this does not affect reactivity. As expected for exposed cysteines, SpyCatcher003 (S49C) will eventually form a disulfide upon storage. This disulfide-bonded form will not be reactive with maleimide or other thiol-reactive reagents. To test the extent of disulfide bond formation, perform SDS-PAGE with Coomassie staining with and without DTT.

  • Theoretical pI: 4.52
  • Extinction coefficient at 280 nm (predicted by ProtParam): 17,420 M-1 cm-1

SpyTag003-MBP:

  • Theoretical pI: 5.87
  • Extinction coefficient at 280 nm (predicted by ProtParam): 69,330 M-1 cm-1

References
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