Part:BBa_K1921013
INPN
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405 - 1000COMPATIBLE 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
Pre-expression
Figure 1.This is the pre-expression using E.coli BL21 at 37 ℃.
Figure 2. This is the pre-expression using E.coli BL21 at 16 ℃.
Figure 3. This is the pre-expression using E.coli BL21 at 25 ℃.
Figure 4.This is the pre-expression using E.coli BL21 induced by different concentration of IPTG.
Figure 5.This is the pre-expression using E.coli BL21 induced by different concentration of IPTG.
Figure 6.This is the pre-expression using E.coli BL21 induced 24h by different concentration of IPTG at 16℃ and 25℃.
Surface display HPLC results
Figure 7. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃.
Figure 8.Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 25℃ with different amount of bacteria.
Figure 9. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced with 0.1mM IPTG for 24h.
Figure 10.Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃ with 0.1mM IPTG for 1h, 4h, 8h, 12h, 16h and 20h.
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