Part:BBa_K4195112

I0500-B0034-rFET-his-B0015

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

Agarose gel electrophoresis (AGE)

When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target (1801 bp).

Fig. 2 The result of regular PCR. Plasmid pSB1C3.

SDS-PAGE

The plasmid verified by sequencing was successfully transformed into E. coli SHuffle T7. After being cultivated and induced by L-arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. After the first time of purification, we found that the protein was poorly expressed, so we did not obtain the target protein. Therefore, we constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into E. coli BL21(DE3). Learn more specific and detailed information in the parts page of this basic part (BBa_K4195009).

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).
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961