Part:BBa_K4614112

_ Encoding sequence of the fusion protein of Wza and SpyCatcher

Wza is an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel α-helical barrel. Mature Wza, the best studied OMA member, is a 359-residue lipoprotein protein that forms SDS-stable octamers. The C terminus of each monomer is exposed on the cell surface, placing the acylated N terminus at the inner leaflet of the outer membrane, consistent with typical outer membrane lipoproteins^[1]. The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag^[2]. [1]Dong C, Beis K, Nesper J, et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein[J]. Nature, 2006,444(7116):226-229. [2]Hatlem, D.; Trunk, T.; Linke, D.; Leo, J.C. Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. Int. J. Mol. Sci. 2019, 20, 2129.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 661
  

Profile

Name:Fusion protein Wza-SpyCatcher
Base Pairs: 1503 bp
Origin: Escherichia coli K12 & Streptococcus pyogenes, synthetic
Properties: Under certain conditions, Wza can display SpyCatcher，which can cross-link with SpyTag, specifically at the poles of the bacteria. cross-link with SpyTag.

Usage and Biology

The SpyTag-SpyCatcher system is widely usedLigation of targeting-antibody with antigen provided a simple route to vaccine generation. SpyRings, from head-to-tail cyclisation, gave major enhancements in enzyme resilience. Linking multiple SpyCatchers gave dendrimers for T-cell activation or Spy networks forming hydrogels for stem cell culture. Synthetic biology applications include integrating amyloid biomaterials with living bacteria, for irreversible derivatisation of biofilms with enzymes or nanoparticles. We also discuss further opportunities to apply and enhance SpyTag/SpyCatcher technology_[3] We try to develop Wza as a new carrier protein, and use its characteristic of being relatively fixed on the cell wall to achieve our special requirements for the spatial location of the display protein. [3]Samuel C Reddington, Mark Howarth,Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher,Current Opinion in Chemical Biology,Volume 29,2015,Pages 94-99,ISSN 1367-5931,https://doi.org/10.1016/j.cbpa.2015.10.002.