This year we supplemented the information of the fusion protein of INPNC. In the last year, TJUsls_China created a protein that combining INPNC and PETase. However, they didn't provided detailed description about this fusion protein. This time we combined INPNC with smURFP (BBa_K2328023 & BBa_K2328032), which is a kind of far red fluorescence protein, because we would like to test the anchoring effect of INPNC with different passenger protein.
Far red fluorescence protein give a better resolution in living imaging because it can reduce background noise/auto-fluorescence caused by cellular materials and solvent (water), improved tissue depth penetration (up to several cm) and high spatial and temporal resolution within limits.
smURFP (small ultra-red FP) is a far red fluorescent protein. It is desirable for our in vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. SmURFP can covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP. Surface display 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. INPNC is an OMP that is found in several plant pathogenic bacteria. 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.
Here we established an approach to display smURFP on the surface of many intestinal bacteria using N-terminal and C-terminal of ice nucleation protein as anchoring motif. In Bacteria cell surface display means we fix the protein onto the out membrane of intestinal bacteria. According to the characteristic that smURFPs are capable to stay at a proper orientation so that they get more possibilities to combine with the biliverdin. Because the highly hydrophilic C-terminal of INP can combine with the out membrane, the display of our passenger protein, smURFP, are allowed to be more stable. Besides, our method solve the problem of visual observation of intestinal bacteria settled in the intestine.
And in this time, we successed in expressing the fusion protein which is about 40.4kDa in the cell membrane in Escherichia coli (E.coil BL21) and prove that this anchoring protein is suitbale for E.coli for surface display construction.
Figure 1. The result of western blot. Lane 1 is E.coli BL21 with no plasmid. Lane 2 is E.coli BL21 with pET28b. Lane 3 is positive control (a protein with His-tag).
 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.
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.
 Haque A, Faizi M S, Rather J A, et al. Next generation NIR fluorophores for tumor imaging and fluorescence-guided surgery: A review. [J]. Bioorganic & Medicinal Chemistry, 2017, 25(7):2017.