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

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.