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INPNC-rLvAPN1

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 display protein of interest on the cell surface (1) .

rLvAPN1

LvAPN1, a protein from the aminopeptidase N family, was identified in Litopenaeus vannamei hemocytes as a receptor for VP_AHPND toxin PirA and PirB, which can help the toxins pass through the cell membrane of hemocytes (2).

rLvAPN1 is a truncated form of LvAPN1 (residues 205-591) that composes of a crystal insecticidal (Cry) toxin binding region and the active site of peptidase-M1 domain, which was reported to directly bind to both PirA and PirB toxins (2). What’s more, there is no glycosylation site in rLvAPN1, which makes it easier to obtain the purified protein by using prokaryotic expression system (such as E. coli).

Usage and design

Engineering 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 (SALAGE), 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-rLvAPN1), rLvAPN1 was fused to the C-terminal of INPNC to surface display for binding PirA and PirB toxins. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then composite part BBa_K4195132 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 PirA and PirB toxins.

Characterization

1.Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (3834 bp) can be observed at the position between 3000 and 5000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195132_pSB1C3.

2. Ability of binding PirA-his and his-PirB on the surface of engineered bacteria

We used BBa_I0500 promoter and RBS (BBa_B0034) to express INPNC- rLvAPN1 protein in E. coli BL21(DE3). The arabinose-induced overnight culture was firstly incubated with purified PirA-his or his-PirB, then FITC-labeled anti-His-tag antibody in turn to verify whether the displayed rLvAPN1 could bind PirA-his and his-PirB or not.

Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria. Purified PirA-his (a) or his-PirB (b) was tested to interact with the displayed receptors (p = 0.0014 for PirA-his, p = 0.0326 for his-PirB).

The ratio of fluorescence intensity (a: λ_Ex = 492 nm, λ_Em = 518 nm; b: λ_Ex = 492 nm, λ_Em = 528 nm; different excitation wavelengths were set at different apparatuses) to OD₆₀₀ of positive control (culture was incubated with PirA-his or 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 rLvAPN1 to PirA-his and his-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_K4195132 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 (3,4). By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed rLvAPN1 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. W. Luangtrakul et al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS Pathog. 17, e1009463 (2021).

3. 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).

4. 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