Part:BBa_K2721024

RFP with SdyTag and SpyCatcher

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 784
  Illegal AgeI site found at 600
  Illegal AgeI site found at 712
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

The covalent nature of Tag-Catcher interaction has facilitated the processing of proteins into various materials forms including all-protein-based, chemically cross-linked hydrogels, functional layer-by-layer thin films, hybrid colloidal assemblies, and living materials[1]. The SpyTag/SpyCatcher, SdyTag/SdyCatcher systems are convenient protein coupling tools for irreversible peptide-protein ligation. According to papers, Spytag forms a covalent isopeptide bound to its protein partner SpyCatcher derived from Streptococcus pyogenes fibronectin-binding protein[2]. Similarly, SdyTag has a 75-fold specificity for its optimized Catcher, named SdyCatcher derived from a related fibronectin-binding protein in Streptococcus dysgalactiae[3].

Fig1 Structure of SpyTag/SpyCatcher.[2]

Considering that the tag-catcher pairs are ideal for binding and immobilization, our team use them as scaffold of protein assembly. Here RFP is a reporter.

Approach and Result

The part was assembled with T7 promoter, RBS and T7 terminator in pET26b+ plasmid vector. E. coli strain BL21(DE3). After growing to an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25℃. We get the protein product via sonication. With N-terminal His-tag, it can be purified by Ni-beads.

Fig 2 Agarose Gel Electrophoresis | M：marker，Lane 1: SnoopCatcher with YFP; Lane 2: RFP with SdyTag and Spycatcher;

Reference

1.J Fang, WB Zhang. Genetically Encoded Peptide-protein Reactive Pairs. Acta Polymerica Sinica 2018 Jan (4):429-444.

2. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7.

3. Tan LL, Hoon SS, Wong FT. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE 11(10): e0165074.

4. P Hengen. Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences, 1995,20(7): 285-286.