AI Article Synopsis

  • This study examines the use of evolved soybean ascorbate peroxidase (APEX2) as a reporter by fusing it to llama nanobodies (single-domain antibodies) to enhance periplasmic expression, which maintains the natural structure of antibodies.
  • The researchers successfully purified significant amounts of these fusion proteins and utilized them in various detection methods like Western blotting and ELISA to visualize viral nucleoproteins.
  • The findings suggest that these sdAb-APEX2 fusions can improve detection of diverse viruses, including a newly discovered virus, and facilitate ongoing studies of various Filovirus species in a controlled laboratory environment.

Article Abstract

We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb; variable domains of heavy chain-only antibodies, VHH) targeted to the periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the genus and cross-reactive within the genus, we show that periplasmic sdAb-APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb-APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti- sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕnglà virus genome. Antibody-antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Mĕnglà NP C-terminal domain. The sdAb-APEX2 fusions also enabled live and detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb-APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521291PMC
http://dx.doi.org/10.3390/v11040364DOI Listing

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