Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mengla virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among Marburgvirus and Dianlovirus.

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 E. coli periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous E. coli outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the Marburgvirus genus and cross-reactive within the Ebolavirus 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 sd

SUBMITTER: Sherwood LJ 

PROVIDER: S-EPMC6521291 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature