Ligation of pET28 region in pGEM plasmid
The recombinant expression region of pET28 was amplified by PCR with the primers pET28 forward (TTTCTGCAGCACCACCCTGAATTGACTCTCT) and pET28 reverse (TTTCTGCAGGGATATAGTTCCTCCTTTCAGCA) flanked by PstI restriction sites (in italic). The forward and reverse primers were localized, respectively, at 383 bases upstream of the T7 promoter and 21 bases downstream of the T7 terminator (Fig. 1A). The pET28 fragment was ligated in the pGEM-T Easy Vector (Promega) following the manufacturer’s instructions. The pGEM-pET28 (Fig. 1B) plasmid was cloned in E. coli Top10, extracted with mini-prep (Wizard® Plus SV Minipreps; Promega), and confirmed by sequencing.
N. caninum culture
The tachyzoite forms of N. caninum Nc-1 isolate were cultured in Vero cell cultures as previously described. The purification of tachyzoites was performed by exclusion chromatography in Sephadex G-25 (PD-10 columns; GE). The recovered parasites were counted in a Neubauer chamber after adequate dilution and applied as the source for RNA extraction.
RNA extraction and cDNA synthesis
RNA from N. caninum was extracted following the Trizol protocol (Invitrogen). Freshly purified tachyzoites (1×107) were solubilized in 1 mL Trizol solution and extracted in 1 mL chloroform, followed by centrifugation at 10,000 g for 15 min. The resulting aqueous phase (200 mL) was replaced into a new tube and the RNA was precipitated with 200 mL absolute isopropanol. RNA was pelleted by centrifugation at 10,000 g for 15 min and washed with 500 mL 75% ethanol. The pellet was air-dried, diluted in 50 mL water and quantified in a 260/280 nm spectrophotometer (GeneQuant Pro; Amersham/GE). cDNA was obtained by reverse transcription following the manufacturer’s instructions (GoScript™ Reverse Transcription System kit). Briefly, 500 ng of RNA was incubated with 5 nM of poli-T primer for 1 h at 37°C. The actin fragment of N. caninum (ToxoDB data bank: NcLIV_003440) was amplified from the 604 to 932 bases with the primers forward NcActBamHI (TTTGGATCCACCACCTCCGCC) and reverse NcActHindIII (TTTAAGCTTCCAATACCCTCGT). The restriction sites BamHI and HindIII are shown in italic. The N. caninum actin fragment was cloned in pGEM-T Easy Vector and sequenced.
Expression of actin fragment in pGEM-pET28 plasmid
Both pGEM (with N. caninum actin fragment) and pGEM-pET28 plasmid were treated with BamHI and HindIII. The NcActin fragment, extracted from pGEM, was ligated in the hybrid plasmid. The ligation was electroporated in E. coli Top10, verified with EcoRI treatment, and sequenced. The pGEM-pET28/Actin was electroporated in E. coli BL21 and submitted to expression inducted by 1 mM IPTG. The bacterial pellets were sonicated with 8 M urea and the recombinant actin fragment was purified in an affinity nickel sepharose column (Ni Sepharose 6 Fast Flow; GE). The extracts and aliquots with the recombinant protein were analyzed by SDS-PAGE and stained with coomassie blue G.
The band corresponding to the recombinant actin was excised and subjected to trypsin digestion. Briefly, the sample was washed 3 times in 25 mM NH4HCO3 pH 8.0 in 50% acetonitrile (ACN) for 15 min each wash, dehydrated in 100% ACN, and dried in a vacuum concentrator (Labconco). The gel was resuspended in 25 ng/µL trypsin solution (Trypsin Gold; Promega), incubated for 16–24 h at 37°C, and peptides extracted with 50% ACN and 5% trifluoroacetic acid (TFA). For MS/MS, the peptide solution was diluted 1:1 in α-cyano-4-hydroxycinnamic acid (10 mg/mL in 50% ACN and 0.1% TFA) and applied in a MALDI plate. The calibrant was a polyethylene glycol (PEG) solution diluted in a matrix (1:1). The identification was performed in a matrix-assisted Laser Desorption/Ionization (MALDI TOF/TOF-Bruker). The data were searched against the N. caninum predicted protein database (www.matrixscience.com), using Mascot software version 2.3.02 (Matrix Science). The carbamidomethylation of cysteine and the oxidation of methionine were set as fixed and variable modifications, respectively.