Abstract:

Objective To analyze the β4-tubulin of Echinococcus granulosus protoscoleces （EgTub4） using bioinformatics tools, and optimize the induction of its soluble expression in Escherichia coli and purify the target protein. Methods The physicochemical properties and structure of EgTub4 protein were analyzed by ProtParam, SMART, Predictprotein and Swiss-model softwares. The signal peptide of EgTub4 sequence was predicted by the online software SignalP 4.1. Total RNA was extracted from E. granulosus protoscoleces and reversely transcribed into cDNA, based on which the EgTub4 gene was amplified by PCR. The recombinant prokaryotic expression vectors pET28a-EgTub4, pET30a-EgTub4 and pET32a-EgTub4 were constructed to explore the optimal induction conditions (including the expression vector, induction temperature, concentration of the inducer isopropyl-β-D-thiogalactoside, induction period, and medium composition) for soluble expression of the recombinant rEgTub4 protein. The recombinant protein was purified using nickel affinity chromatography, isolated by SDS-PAGE, and analyzed by Western blotting. 　Results  As predicted by bioinformatics analysis, the EgTub4 protein had a pI of 4.79 and a Mr of 49 700, did not contain any signal peptide, but had three domains: a GTPase domain, a C-terminal, and a coiled coil region. Double digestion and sequencing results confirmed the successful construction of the three recombinant plasmids(pET28a-EgTub4, pET30a-EgTub4 and pET32a-EgTub4). Western blotting analysis showed that the Mr values of recombinant protein in the expression vectors pET28a (+), pET30a (+) and pET32a (+) were 56 000, 60 000 and 68 000, respectively. The expression of EgTub4 gene in pET28a(+) was insoluble, mainly in the form of inclusion body, which needs to be purified under denaturing condition. The expression of EgTub4 gene in pET30a (+) and pET32a (+) was partially soluble, which can be purified under native conditions. In this study, pET30a (+) was selected as the expression vector of EgTub4 gene. The optimum induction conditions for soluble expression of target protein were: T = 25 ℃, [IPTG] = 0.01 mmol/L, t = 10 h, 2 × YT. The optimum concentration of imidazole for removing other protein was 150 mmol/L. The best elution concentration of imidazole was 500 mmol/L. Western blotting showed that the recombinant protein rEgTub4 could be specifically recognized by His-Tag and β-tubulin monoclonal antibody, resulting in a specific band at the Mr been 60 000.  Conclusion This study provides comprehensive bioinformatics prediction and analysis of EgTub4 which has been cloned, expressed and purified. 
  

Key words: Echinococcus granulosus, Protoscole, β4-tubulin, Prokaryotic expression, Solubility, Bioinformatics