Recombinant Expression of Toxoplasma and Plant Chaperone Proteins

A peptide of Toxoplasma gondii SAG1 (SAG1_HC) (from residue 221 to residue 319 of the protein) that encodes both T- and B-cell epitopes [10 (link)] was amplified by PCR using the plasmid pRSET-A-SAG1_77–322 as a template [21 (link)]. The forward primer sequence was 5′-ggt acc ATA AAG TTC CTC AAG ACA AC-3′ and the reverse primer sequence was 5′-aag ctt CTA AAT GGA AAC GTG ACT GGC-3′ flanked by KpnI and HindIII restriction sites (lowercase), respectively. The N. benthamiana Hsp90.3 full length sequence (NbHsp90.3) was amplified by PCR using the plasmid pRSET-A-NbHsp90.3 as a template [18 (link)]. The forward degenerated primer sequence was 5′-gga tcc ATG GCG GAS GCA GAR ACS TTT GCW TTY CAA GC-3′ and the reverse primer sequence was 5′-ctc gag GTC TAC TTC CTC CAT CTT TTC AGC ATC ATC AGC-3′ flanked by BamHI and XhoI restriction sites (lowercase), respectively. The PCR products were first cloned separately into pGEM-T easy vectors (Promega, Fitchburg, WI, USA). After sequencing, the isolated segments were sequentially cloned into pRSET-A (Invitrogen, Carlsbad, CA, USA) to construct pRSET-A-NbHsp90.3-SAG1_HC expression vector.