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

Area of Science:

• Molecular Biology
• Parasitology
• Biochemistry

Background:

• Echinococcosis poses a significant public health challenge, with benzimidazoles as the primary treatment.
• Tubulin proteins are the suspected targets of benzimidazole drugs used against echinococcosis.

Purpose of the Study:

• To characterize the α- and β-tubulin isoforms from Echinococcus granulosus.
• To provide recombinant tubulin proteins for screening anti-parasitic compounds.

Main Methods:

• Cloning of full-length cDNAs for E. granulosus α9- and β4-tubulin using reverse transcription PCR.
• Recombinant expression in E. coli, purification via nickel affinity chromatography, and protein refolding.
• Monitoring tubulin polymerization using UV spectrophotometry, confocal microscopy, and transmission electron microscopy (TEM).

Main Results:

• Nucleotide sequence analysis confirmed the lengths of α9-tubulin (1356 bp) and β4-tubulin (1332 bp) cDNA.
• Recombinant α9- and β4-tubulin proteins (451 and 443 amino acids, respectively) were successfully expressed and purified with average yields of 2.0-3.0 mg/L and 3.5-5.0 mg/L.
• Purified recombinant tubulins demonstrated polymerization into microtubule-like structures in vitro.

Conclusions:

• The study successfully produced functional recombinant Echinococcus granulosus α9- and β4-tubulin.
• These recombinant tubulins are valuable tools for developing novel therapeutic strategies against echinococcosis by enabling the screening of targeted compounds.