Recombinant α- and β-tubulin from Echinococcus granulosus: expression, purification and polymerization

Abstract

Echinococcosis, which causes a high disease burden and is of great public health significance, is caused by the larval stage of Echinococcus species. It has been suggested that tubulin is the target of benzimidazoles, the only drugs for the treatment of echinococcosis. This study evaluated the characteristics of tubulins from Echinococcus granulosus. The full-length cDNAs of E. granulosus α- and β-tubulin isoforms were cloned by reverse transcription PCR from protoscolex RNA. Then, these two tubulin isoforms (α9 and β4) were recombinantly expressed as insoluble inclusion bodies in Escherichia coli. Nickel affinity chromatography was used to purify and refold the contents of these inclusion bodies as active proteins. The polymerization of tubulins was monitored by UV spectrophotometry (A350) and confirmed by confocal microscopy and transmission electron microscopy (TEM). Nucleotide sequence analysis revealed that E. granulosus 1356 bp α9-tubulin and 1332 bp β4-tubulin encode corresponding proteins of 451 and 443 amino acids. The average yields of α9- and β4-tubulin were 2.0–3.0 mg/L and 3.5–5.0 mg/L of culture, respectively. Moreover, recombinant α9- and β4-tubulin were capable of polymerizing into microtubule-like structures under appropriate conditions in vitro. These recombinant tubulins could be helpful for screening anti-Echinococcus compounds targeting the tubulins of E. granulosus.

Characterization of the B-Cell Epitopes of Echinococcus granulosus Histones H4 and H2A Recognized by Sera From Patients With Liver Cysts

“…However, the recombinant or synthetic antigenic proteins assayed have less sensitivity and specificity than ex vivo parasite antigens and/or predictive values have not been conclusive. These are the cases of AgB ( Hernández-González et al., 2008 ; Savardashtaki et al., 2017 ; Han et al., 2019 ; Salah et al., 2021 ), Ag5 ( Barbieri et al., 1998 ), EPC1-calcium binding protein from E. granulosus protoscoleces ( Fathi et al., 2016 ), and recombinant tubulins obtained, but not clinical studies with these antigens have been performed ( Liu et al., 2018 ).…”