Part:BBa_K4195024

Biology

INPNC

INPNC is a truncated form of ice nucleation protein (INP) consisting of N- and C-terminal domains. It is a membrane protein commonly used to target protein onto the cell membrane (1).

rFET

rFET is a truncated form of the A chain of mouse fetuin-B (residues 141-169). Vertebrate fetuins are multi-domain plasma-proteins of the cystatin-superfamily (2). It was reported that mouse fetuin-B shows high inhibition effect to the toxin PirB (3). We used the ClusPro (4) to evaluate the affinity of mouse fetuin-B to PirA and PirB. The results showed that the 141-169 residues of the A chain of mouse fetuin-B have higher affinity to PirA and PirB than the complete A chain of mouse fetuin-B. What’s more, there is no glycosylation site in rFET sequence, so the expression of recombinant rFET by engineered E. coli can be available and functional. In summary, we chose the 141-169 residues of the A chain of mouse fetuin-B as the functional inhibitor and named it rFET (BBa_K4195009).
The rFET can be displayed on the surface of the engineered bacteria and OMVs (outer membrane vesicles) due to the localization of INPNC. The OMVs with rFET displayed is more stable in the environment than rFET and is a better choice for binding to toxins.

Usage and design

Engineering outer membrane vesicles (OMVs) for treating and preventing AHPND caused by the pathogen V. parahaemolyticus are a significant part of OMEGA project (Operable Magic to Efficiently Getting over AHPND). Based on the efforts of our previous projects in 2020 (AnTea-Glyphosate) and 2021 (SALVAGE), we further developed the surface display system on the OMVs released by the engineered bacteria. The usage of cargo proteins was no more limited to enzymes that are usually utilized to catalyze series bio-chemical reactions, since some receptors or ligands involved in complex protein-protein interaction (PPI) were selected as the cargo candidates. This year, we chose two classic anchor proteins, ClyA and INPNC, to construct the display cassette with various cargo proteins including rFET (receptor), rLvAPN1 (receptor), TTPA (ligand) and TTPB (ligand) (Fig. 1). On one hand, with the receptors displayed, OMVs will gain the function of neutralizing toxins secreted by V. parahaemolyticus. On the other hand, with the assistance of ligands displayed on the surface, OMVs will become a special vector to deliver antimicrobials for the specific pathogen. In summary, we have taken a step closer to the collections of extracellular functional elements (EFE), combining the OMVs, secretion systems and surface display systems which we have been dedicated to since 2020. Learn more information from our Design page.

Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.
For this part (INPNC-rFET), rFET was fused to the C-terminal of INPNC to surface display for neutralizing toxins PirA and PirB. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then composite part BBa_K4195125 was obtained. We transformed the constructed plasmid into E. coli BL21(DE3) for further verification of its expression and function on the surface of E. coli and OMVs, including the interaction between rFET and PirA or PirB.

Characterization

1.Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (2484 bp) can be observed at the position between 2000 bp and 3000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195125_pSB1C3.

2. Ability of binding PirB on the surface of engineered bacteria

We used BBa_I0500 promoter and RBS (BBa_B0034) to express INPNC-rFET protein in E. coli BL21(DE3). The arabinose-induced overnight culture was then incubated with purified his-PirB and FITC-labeled anti-His-tag antibody in turn to verify whether the displayed rFET could bind his-PirB or not.

Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria (p = 0.0426).
The ratio of fluorescence intensity (λ_Ex = 492 nm, λ_Em = 518 nm) to OD₆₀₀ of positive control (culture was incubated with his-PirB) is higher than that of negative control (culture was incubated with 1×TBST) (Fig. 3), which indicates that our surface display system works well and the binding ability of rFET to PirB is retained on the surface of bacteria.

3. Ability of binding toxins on the surface of OMVs

For the test on OMVs, the OMVs were firstly extracted from the culture of engineered bacteria harboring BBa_K4195125 after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified PirA-his or his-PirB and anti-His-tag antibody in turn, and finally probed by the HRP-conjugated secondary antibody. By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins (5, 6), we could characterize whether the displayed rFET on OMVs is functional or not.

Reference

1. E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol. 29, 79-86 (2011).
2. K. Karmilin et al., Mammalian plasma fetuin-B is a selective inhibitor of ovastacin and meprin metalloproteinases. Sci. Rep. 9, 546 (2019).
3. M. Victorio-De Los Santos et al., The B Subunit of PirAB^vpToxin Secreted from Vibrio parahaemolyticus Causing AHPND Is an Amino Sugar Specific Lectin. Pathogens. 9, 182 (2020).
4. D. Kozakov et al., The ClusPro web server for protein-protein docking. Nat. Protoc. 12, 255-278 (2017).
5. J. L. Valentine et al., Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies. Cell Chem. Biol. 23, 655-665 (2016).
6. T. C. Stevenson et al., Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies. Proc. Natl. Acad. Sci. U. S. A. 115, E3106-E3115 (2018).

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 405
  Illegal NgoMIV site found at 964
  Illegal AgeI site found at 507
  Illegal AgeI site found at 823
  
• 1000
  COMPATIBLE WITH RFC[1000]