Part:BBa_K1921016

PETase+INPNC

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 861
  Illegal NgoMIV site found at 1194
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

This part is a fusion protein of the ice nucleation protein without the central part and PETase. PETase is a new protein enzyme found in bacteria which can decompose PET. Through this method, we can anchor PETase on the out membrane of Escherichia coli, then we can use this typical Escherichia coli to decompose PET. This is a way called whole cell catalysis. Using this method, we don’t need to purify the protein. In addition, prokaryote surface display system method is mature enough. INP is frequently used to develop whole-cell biocatalysts, primarily because: (i) INP does not appear to be hampered by the size of the passenger; and (ii) INP is compatible with the translocation and surface display of proteins that contain multiple cofactors as well as disulfide-bond-containing passengers.

Biology

PETase was found from a kind of microorganism(Ideonella sakaiensis 201-F6) living on PET as the main carbon source. It can degrade macromolecular polymers into monomers. PETase is the only enzyme found in bacteria which can degrade PET.
Surface expression of recombinant proteins was first described more than 30 years ago.Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. INP is an OMP that is found in several plant pathogenic bacteria. Our inaK is from Pseudomonas. INP has several unique structural and functional features that make it highly suitable for use in a bacterial surface display system. The specific amino acids of the N-terminal domain are relatively hydrophobic and link the protein to the OM via a glycosylphosphatidylinositol anchor. The C-terminal domain of the protein is highly hydrophilic and exposed to the medium. The central part of INP comprises a series of repeating domains that act as templates for ice crystal formation. However, the N-terminal domain appears to be the only prerequisite for successful targeting and surface-anchoring. With the C-terminal of INP the anchoring of PETase can show higher stability.

Reference

[1] Shosuke, Yoshida, 1, 2*, Kazumi, Hiraga, 1, Toshihiko, Takehana, 3, Ikuo, Taniguchi, 4, Hironao, Yamaji, 1, Yasuhito, Maeda, 5, Kiyotsuna, Toyohara, 5, Kenji, Miyamoto, 2†, Yoshiharu, Kimura, 4, Kohei, Oda1. A bacterium that degrades and assimilates poly(ethylene terephthalate)[J]. SCIENCE, 2016: 1196-1199
[2]Edwin, van, Bloois1, Remko, T, Winter1, Harald, Kolmar2, and, Marco, W, Fraaije. Decorating microbes: surface display of proteins on Escherichia coli[J]. CELL Press, 2011, 29(2): 79-86