Difference between revisions of "Part:BBa K1921013"
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===Usage===
 
===Usage===
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The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. AIDA is one of the members of autotransporter family. We call it one of an anchor proteins for it can immobilize on the outer membrane of Escherichia coli. It is very useful because we can insert other proteins’ sequence into the AIDA sequence, and then the protein will be immobilized on the outer membrane. We can use it to do whole cell catalysis. In addition, it also have a broad range of applications in molecular biology, biochemistry, biotechnology, microbiology and vaccinology . Today we use this part to display our PETase on E.coli’s surface. Compared with the eukaryotic surface display system, display system with surface expression in prokaryotes cycle is short. In addition, prokaryote surface display system method is simple and mature. AIDAc is the autotransporter adhesin involved in diffuse adherence, so it can anchor stably on the membrane.  
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This part is the N-terminal domain of the ice nucleation protein. Here we established an approach to display PETase on the surface of Escherichia coli (E. coli) using N-terminal of ice nucleation protein as anchoring motif. Compared with the other anchoring motif, INP can be expressed at the cell surface of E. coli at a very high level, without affecting cell viability Bacteria cell surface display means we fix the enzyme onto the out membrane of E.coli. According to the immobilization the enzyme are capable to stay at a proper orientation so that they get more possibilities to combine with the PET. Besides, our method solve the problem of the degradation PETase. The enzyme will be stable in the cell surface display system.
  
 
===Biology===
 
===Biology===
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Surface expression of recombinant proteins was first described more than 30 years ago. AIDA is one of an anchor proteins which belongs to Escherichia coli(Escherichia coli strain 2787). We find its sequence from NCBI. Surface display using AIDA contains three parts: signal peptide, passenger domain and anchor protein AIDAc. So this is a C-terminal anchoring. AIDAc protein structure has been analyzed. It is a transmembrane protein across the 12 membrane and its shape is just like a β-barrel. Theβ-barrel can be anchored on the outer membrane so that the special protein can be displayed on the surface.
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Surface expression of recombinant proteins was first described more than 30 years ago.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.
  
 
===Reference===
 
===Reference===
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[1] Jarmander, Johan; Gustavsson, Martin; Thi-Huyen Do: A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli. MICROBIAL CELL FACTORIES 2012,11
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[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
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[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

Revision as of 14:40, 12 October 2016


INPN


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 72
    Illegal NgoMIV site found at 405
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

This part is the N-terminal domain of the ice nucleation protein. Here we established an approach to display PETase on the surface of Escherichia coli (E. coli) using N-terminal of ice nucleation protein as anchoring motif. Compared with the other anchoring motif, INP can be expressed at the cell surface of E. coli at a very high level, without affecting cell viability Bacteria cell surface display means we fix the enzyme onto the out membrane of E.coli. According to the immobilization the enzyme are capable to stay at a proper orientation so that they get more possibilities to combine with the PET. Besides, our method solve the problem of the degradation PETase. The enzyme will be stable in the cell surface display system.

Biology

Surface expression of recombinant proteins was first described more than 30 years ago.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.

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