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.


Introduction
Cystic echinococcosis, which is a global health issue that affects humans and animals, is caused by the metacestode larval stage of Echinococcus granulosus [23]. The [3,4]. In livestock and humans, these parasites are mainly located in the liver and lungs [4]. Mebendazole and albendazole, both benzimidazoles (BZs), are drugs for the therapy of *Corresponding authors: zhanghaobing2@163.com echinococcosis [7,28]. Circumstantial evidence suggests that BZs suppress the polymerization of parasite microtubules (MTs) by binding to the b-tubulin [1,17], which has made tubulin an attractive target for drug development [27,39], but studies related to the MTs of E. granulosus have been limited.
Microtubules are highly dynamic structures that perform diverse and critical functions in cell structure, cell division, motility, and signal transduction [5,8]. MTs are composed of soluble tubulin subunits comprising aand b-tubulins, which are similar in mass (~55 kDa) and share approximately 40% amino acid identity. The formation of MTs reflects the balance between polymerization and de-polymerization of a/b-tubulin heterodimers. The tubulin polymerization assay has already been a powerful tool in characterizing the interactions between drugs and MTs. To date, most functional analyses of MTs have used native tubulins purified from mammalian brain, eukaryotic organisms, kinetoplastid parasites (Leishmania, Trypanosoma) and Saccharomyces cerevisiae [30]. Although abundant tubulin can be isolated from these sources, the purified proteins are composed of multiple tubulin isoforms and contain only those tubulin subpopulations with assembly competency [35,37]. Moreover, these results are affected by the other proteins and cofactors that co-purify with native tubulins [27]. In addition, due to the difficulty in collecting enough E. granulosus for tubulin extraction, this simple and rapid purification method is not applicable in E. granulosus or E. multilocularis, which hinders the study of the MTs of this parasite.
Fortunately, there are reports on recombinant human tubulins [37] and helminth tubulins [20,26] that could polymerize into MTs, indicating that recombinant MTs could be used for high-throughput screening. Hence, based on the previously reported tubulin genes of the parasite and the methods for expressing tubulin and determining the polymerization of the a/b-tubulin heterodimer, we conducted a study on the characteristics of E. granulosus tubulin genes and polymerization.
In this study, aand b-tubulin of E. granulosus were expressed in Escherichia coli and purified, and these heterodimers were shown to polymerize into microtubule-like structures.

RNA isolation and cDNA synthesis
Protoscoleces were isolated from cysts in the liver of sheep infected with E. granulosus (G1 strain, Qinghai, China). Then, total RNA was extracted with the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions, followed by reverse transcription using a first-strand cDNA synthesis kit (Toyobo, Japan). The genes encoding E. granulosus a 9 -and b 4 -tubulin were amplified with ExTaq DNA polymerase (Takara, Japan) using gene-specific primers. For a 9 -tubulin, the forward primer was 5 0 -CGCGAGCTCATGCGTGAATGTATCAGTAT-3 0 with a Sac I restriction site (in bold), and the reverse primer was 5 0 -AGCGGCCGCTTAGTACTCCTCGCCCTCTT-3 0 with a Not I restriction site (in bold). For b 4 -tubulin, 5 0 -CGCGG-ATCCATGCGAGAGATAGTACACGTT-3 0 and 5 0 -CCCAA-GCTTTTATGCTTCTTCCTCT-3 0 were used as the forward and reverse primers, containing BamH I and Hind III restriction sites (in bold), respectively. The PCR reaction mixture contained 1 lM each primer, 200 lM dNTP mixture, 1· PCR buffer and 0.5 units ExTaq DNA polymerase. PCR conditions were as follows: 5 min at 95°C for denaturation; 35 cycles of amplification (40 s at 95°C, 30 s at 60°C/57°C for a 9 -tubulin/b 4 -tubulin, 90 s at 72°C); 10 min at 72°C for extension. PCR products were separated on 1.2% agarose gels and purified with the Gel Extraction Kit (Qiagen, USA).

Expression of recombinant proteins
The purified PCR fragments were directly cloned into the pMD19-T vector (Takara, Japan) for TA cloning using the Mighty TA-Cloning Kit (Takara, Japan) and transformed into competent Escherichia coli DH5a cells (Tiangen, China), which were incubated at 37°C overnight on a Luria-Bertani (LB) plate containing 100 lg/mL ampicillin (Sigma, USA). A single clone from each construct was selected and sequenced to ensure sequence fidelity. The verified a 9 -and b 4 -tubulin sequences were cut from the pMD19-T construct by double enzyme digestion and directionally ligated into the pET30a(+) vector (Novagen, USA), which had previously been digested with the same enzymes. Then, plasmid constructs (pET30aa 9 and pET30a-b 4 ) were confirmed by double enzyme digestion with corresponding enzymes.
The pET30a-a 9 and pET30a-b 4 were finally transformed into competent BL21 (DE3) cells (Tiangen, China) using the heat shock method. The positive clones were selected and cultured in 2 L LB medium containing 50 lg/mL kanamycin until the mid-log phase. Expression was induced with 1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) for 6 h at 37°C/200 rpm. The cells were harvested at 8000 · g for 15 min, and the pellet was washed with phosphate buffer saline (PBS). The cells were centrifuged again and resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 0.5 mM PMSF, 0.1% Triton X-100, pH 7.4), disrupted by sonication. The inclusion bodies were collected by centrifugation at 12,000 · g, 4°C for 20 min.

Purification of recombinant proteins
The inclusion bodies were dissolved in binding buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 8 M urea, pH 7.4), collected by centrifugation at 12,000·g for 20 min at 4°C and loaded onto an Ni 2+ Sepharose column (GE Healthcare, USA) pre-equilibrated with the binding buffer. The column was subsequently washed with five column volumes of binding buffer, followed by washing buffer with a linear gradient of urea ranging from 8 M to 0 M. The refolded fusion protein was eluted with elution buffer (50 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 7.4) and concentrated in an Amicon Ultra centrifugal filter (Millipore, USA). The concentration of recombinant protein was evaluated using a Bradford Kit (Tiangen, China).

Western blotting
The purified a 9 -or b 4 -tubulin protein was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using anti-His antibody (CST, USA, #2366), anti-a-tubulin antibody (CST, USA, #3873) and anti-b-tubulin antibody (CST, USA, #2128) as primary antibodies; the PVDF membrane was blocked for 1 h and then incubated with a 1/1000 dilution of primary antibody at 4°C for 4 h. The membranes were washed and then incubated with a 1/2500 dilution of anti-mouse (CST, USA, #7076) or anti-rabbit IgG antibody conjugated with HRP (CST, USA, #7074) as a secondary antibody at 4°C overnight and then washed again. The ECL Kit (Tanon, China) was used to detect the proteins on the PVDF membrane.

Tubulin polymerization assay
Known concentrations of a 9 -and b 4 -tubulin were diluted with G-PEM buffer (80 mM PIPES, 2 mM MgCl 2 , 0.5 mM EGTA, 1 mM GTP, pH 6.9) to yield final tubulin concentrations of 0.25, 0.5, 1, 2, 3, and 4 mg/mL in a 40 lL reaction mixture. The reaction was carried out at 37°C, and the OD value was measured at 350 nm every 30 s in a Synergy2 spectrophotometer (Biotek, USA).

Immunofluorescence and confocal microscopy
The mixture of 2 mg/mL a 9 -and b 4 -tubulin was allowed to polymerize for 1 h at 37°C and centrifuged at 12,000·g to collect polymerized tubulins. The samples were washed with PBS and centrifuged again. The pellet was fixed in 4% paraformaldehyde at room temperature for 1 h. After washing with PBS five times, the polymerized tubulins were blocked for 1 h at room temperature and incubated with a 1/150 dilution of mouse anti-a-tubulin/Alexa-Fluor 488 antibody (CST, USA, #8058) or anti-b-tubulin/Alexa-Fluor 647 antibody (CST, USA, #3624) at 4°C overnight. Then, the samples were washed with TBST (1· TBS, 0.1% Tween 20) five times before observation under an A1R-si confocal microscope (Nikon, Japan).

Transmission electron microscopy (TEM)
Polymerized tubulins were collected for TEM analyses according to the method reported by Vulevic and Correia [38]. In brief, samples were suspended in 100 lL of PEM buffer (80 mM PIPES, 2 mM MgCl 2 , 0.5 mM EGTA, pH 6.9). A total of 30 lL polymerized samples were diluted with 10 lL of 0.4% glutaraldehyde for 1 min at room temperature, and 10 lL of tubulin solution was applied to a 200-mesh, copper/formvar coated grid for 1 min, washed using dH 2 O and stained for 10 min using 1% uranyl acetate. Finally, samples were air dried and viewed with a Tecnai G2 Spirit transmission electron microscope (FEI, USA).

Results
Amplification of the a 9 -and b 4 -tubulin genes and plasmid construction Echinococcus granulosus full-length a 9 -and b 4 -tubulin cDNAs were amplified; the amplicons contained 1356 bp and 1332 bp coding regions for a 9 -and b 4 -tubulin, respectively (Supplementary Table S1). The a 9 -and b 4 -tubulin genes were predicted to encode proteins with 451 and 443 amino acids, and the theoretical molecular masses were 50.17 kDa and 49.70 kDa (Figs. 1-3).

Analyses of a 9 -and b 4 -tubulin sequences
The sequences of E. granulosus a 9 -and b 4 -tubulin were compared with other aand b-tubulins from different organisms, which showed high degrees of homology (Figs. 2 and 3), especially in some highly conserved domains. As shown in Figure 2, the conserved tubulin acetylation site K40 was also found in E. granulosus a 9 -tubulin and the a-tubulins of humans, Hymenolepis microstoma, Haemonchus contortus, Schistosoma japonicum, and Toxoplasma gondii. The potential GTP-binding site in E. granulosus a 9 -tubulin was present at residues 140-146 (Figs. 2 and 4). In addition, the RGD motif, serving as a cell attachment sequence, was located at residues 320-322. A tyrosine is conserved in highly divergent C-terminal sequences and is involved in the post-translation modifications (PTMs) of tyrosination/detyrosination. Sequence alignment of E. granulosus b 4 -tubulin and b tubulins from other organisms indicated that E. granulosus b 4 -tubulin had conserved His6, Tyr50, Asn165, Ph167, Glu198, Tyr200, and Arg241 (Fig. 3). The potential GDP-binding site in E. granulosus b 4 -tubulin located at residues 138-146 was highly conserved in all groups (Figs. 3 and 4).
Expression and purification of recombinant a 9 -and b 4 -tubulin Recombinant a 9 /b 4 -tubulin was overexpressed mainly in inclusion bodies when E. coli BL21 (DE3) was induced with 1 mM IPTG. The purification yields of a 9 -and b 4 -tubulin were 2.0-3.0 mg/L and 3.5-5.0 mg/L of cell culture, respectively. Single protein bands with the expected molecular weight of a 9 -tubulin or b 4 -tubulin were found on SDS-PAGE gels (Fig. 1a). Furthermore, the recombinant protein was specifically recognized by commercial anti-His antibody, anti-a-tubulin antibody, and anti-b-tubulin antibody, which confirmed the successful expression of recombinant protein (Fig. 1b). The native a/btubulin of the E. granulosus protoscolex, which was in extremely low concentrations, was detected in Western blots by commercial anti-a-and anti-b-tubulin antibodies (Fig. 1b).

Polymerization of recombinant a 9 -and b 4 -tubulin
In this study, continuous A 350 was recorded during polymerization of tubulin at different concentrations. An increase in absorbance was observed for the first 13-43 min, followed by a short initial lag period and a gradual levelling off (Fig. 5a). The optimum concentration of tubulin for polymerization was 2 mg/mL, and the curve is a typical polymerization curve that contains the nucleation, growth, and steady-state equilibrium phases of MT polymerization. By immunofluorescence, recombinant a 9 -tubulin and b 4 -tubulin were detected in polymerized tubulins (Fig. 5b), suggesting that these two tubulin isoforms could polymerize with each other under the proper conditions. In addition, the formation of a microtubule-like structure observed by electron microscopy again proved the polymerization of the tubulins (Fig. 5c).

Discussion
MTs, which are built from a/b-tubulin heterodimers, play an important role in nearly all cellular and developmental processes of eukaryotic cells [23]. The E. granulosus a 9 -and b 4 -tubulin sequences reported in this study were retrieved from the publicly available E. granulosus genome data in the Sanger Database (https://www.sanger.ac.uk/resources/downloads/helminths/echinococcus-granulosus.html). There are 14 proteins denoted as a-tubulin and 10 proteins denoted as b-tubulin in the genome data. We denominated these as E. granulosus a 1 -to a 14 -tubulin and b 1 -to b 10 -tubulin and analyzed the transcript levels of these sequences by real-time PCR. In both the cyst and protoscolex, a 9 -tubulin and b 4 -tubulin were highly expressed (unpublished data). Hence, a 9 -and b 4 -tubulins were selected for subsequent studies.  Previous analyses of tubulin sequences indicated that tubulins are generally highly conserved among species, but the C-termini are highly divergent [6]. Unsurprisingly, some conserved sites and domains were also found in E. granulosus a 9 -and b 4 -tubulin, such as the acetylation sites [16], GTPbinding sites and the RGD sequence [11]. As the best-characterized acetylation site on tubulin, K40 was also conserved in a 9 -tubulin. Many studies have shown that MT acetylation is not necessary for cell survival [9] and is considered to be a marker of MT stability [31]. At present, the acetylation of MTs has mainly been studied in protozoans, and it was concluded that K40 acetylation stabilizes MTs and is required for parasite replication [36], but no data are available for E. granulosus or E. multilocularis. Moreover, the highly divergent C-terminal domain of tubulin is related to tubulin polymerization and interactions with other factors and proteins. The C-terminal sequence of E. granulosus a 9 -tubulin was not fully conserved from other a-tubulins, but the last conserved tyrosine residue indicated that E. granulosus a 9 -tubulin can undergo enzymatic removal and re-addition as part of a detyrosination/tyrosination cycle [41], which affects microtubule-associated proteins (MAPs) that function in a wide range of biological processes [25]. In this study, E. coli was used to produce sufficient amounts of tubulins for MT polymerization experiments, but the shortcoming of this expression system is the lack of protein modification. Therefore, the subsequent study of tubulin modification will require the use of a eukaryotic expression system.
In addition to the modification site, the drug binding sites of b-tubulin are of interest. Mutations at positions 6,50,165,167,198,200 and 241 are related to benzimidazole resistance in parasites, fungi, and plants [2,15,29]. The sequence alignment indicated that His6, Tyr50, and Glu198 are conserved in most tubulins, as shown in Figure 3. In H. contortus, the F200Y mutation is most often related to the resistance profile. Specifically, helminths susceptible to benzimidazole present Phe at position 200; thus, replacing Phe with Tyr may confer the resistant phenotype [14]. Until now, no BZ resistance in Echinococcus spp. has been reported, but the reported analyses of E. multilocularis tubulin sequences predicted sensitivity of EmTub-1 and Em Tub-3 and low binding affinity of