Part:BBa_K1989054

Triplespytag with MBP and His-tag

Usage and Biology

In the last few years, hydrogens made from natural or synthetic polymers have been investigated due to their extensive application in clinical medicine and synthetic biology. Compared to traditional biological material, protein-based multifunctional biological material is low-cost, facile and eco-friendly. However, strategies for assembling 3D molecular networks synthesized only by protein molecular remain underdeveloped. The reason why investigating this technology is still tough is lack of protein-based cross linking agents. Inspired by the self-catalysis of isopeptide bond between Lys and Asp in Streptococcus pyogenes fibronectin-binding protein FbaB, researchers split the catalytic domain and obtained two peptide called SpyTag(the short one) and SpyCatcher(the long one) which are able to form isopeptide bond with the other without any assistant. By fusing SpyTag and SpyCatcher with functional domains respectively, researchers solved the problem tactfully. In order to using SpyTag and SpyCatcher system as scaffold, we fused three SpyTag spaced by (VPGVG)4 with 6xHistag in N-terminal and another functional protein called Mercury-binding Protein(MBP) in C-terminal.

Based on our results, the fused protein Histag-triple spytag-MBP possess both isopeptide bond forming function and mercury-binding ability . Thus, using Histag-3A-MBP as a part of hydrogel formation, we can obtain our multifunctional biomaterial.

References

1. Rodolphe Barrangou, Christophe Fremaux, Hélène Deveau, et al. CRISPR provides acquired resistance against viruses. Science, 2007, 315: 1709-1712.

2. Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011;471:602–7.

3. Martin Jinek, Krzysztof Chylinski, Ines Fonfara, et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012, 337: 816-821.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 578
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]