Part:BBa_K4701205

SpyTag for T7 systems (with C-terminus linker)

The FbaB protein found in S. pyogenes contains a CnaB2 domain with a highly stable isopeptide bond [1]. This domain has been split into two fragments and engineered, the larger of the two called SpyCatcher spontaneously forms an isopeptide bond with the smaller fragment called SpyTag. This allows for the creation of fusion proteins.

This SpyTag sequence is for use with E. coli, and more specifically T7 based expression systems when the SpyTag is located on the N-terminus of the protein of interest [2]. This sequence is recommended in such designs as commonly used SpyTag sequences can cause secondary structures near the Shine-Dalgarno sequence of the T7 promoter, reducing transcription efficiency.

References

[1] Zakeri, B., et al. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. https://doi.org/10.1073/pnas.1115485109

[2] Keeble, A. H., & Howarth, M. (2019). Insider information on successful covalent protein coupling with help from SpyBank. In Methods in Enzymology (Vol. 617, pp. 443–461). Academic Press Inc. https://doi.org/10.1016/bs.mie.2018.12.010

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]