Part:BBa_K4195009

Biology

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 (1). It was reported that mouse fetuin-B shows high inhibition effect to the toxin PirB (2). We used ClusPro (3) to evaluate the affinity of mouse fetuin-B to PirA and PirB. The result showed that the 141-169 residues of the A chain of mouse fetuin-B has 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.

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 Designpage.

Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.
For this part (rFET-his), a His-tag (6×His) was fused to the C-terminal of rFET to purify the recombinant protein. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then composite part BBa_K4195112 was obtained. We transformed the constructed plasmid into E. coli SHuffle T7 for protein purification.

Characterization

Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (114 bp) can be observed at the position around 100 bp and 250 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195009_pET-28a(+).

Reference

1. K. Karmilin et al., Mammalian plasma fetuin-B is a selective inhibitor of ovastacin and meprin metalloproteinases. Sci. Rep. 9, 546 (2019).
2. 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).
3. D. Kozakov et al., The ClusPro web server for protein-protein docking. Nat. Protoc. 12, 255-278 (2017).