Snab 1

Restriction enzymes SnaB I and Bsp 1407I (TaKaRa Bio Inc (Japan)) were used to replace the gVII of the helper plasmid with the SBP minigene. Five microliters of ligation product was then transformed into a freshly prepared chemically competent DH5αF′ bacterial strain, by subjecting the bacteria to a heat shock at 42 °C for 30 s, after which they were plated on LB agar (Invitrogen, Ghent, Belgium) plates with 15 μg/ml of the antibiotic Chlr and incubated at 37 °C overnight. A negative control was also prepared where the DH5αF′ bacterial strain was transformed with unmodified M13cp. DH5αF′ cells already containing the Chlr-resistant M13cpSBP or M13cp helper plasmid were prepared to be competent and cotransformed with Amp-resistant phagemid pspB, either empty (pspB) or bearing the cDNA of the autoantigenic target RA21 fused to its pVI gene (pspB RA21), in the same way as with the helper plasmid. The newly cotransformed DH5αF′ colonies were plated in 2×YT media (BD (Erembodegem, Belgium)) containing 15 μg/ml of Chlr and 100 μg/ml of Amp.

Restriction enzymes Sac I, SnaB I and Not I were purchased from Takara Bio Inc., Japan, and used as detailed by the manufacturer. Yeast nitrogen base w/o amino acids (YNB), D-sorbitol, Kanamycin monosulfate, Zeocin and geneticin sulfate (G-418S) were obtained from Solarbio, Beijing, China. Oxoid™ peptone and yeast extract were purchased from Thermo Scientific, Germany.
P. pastoris was grown in yeast peptone dextrose medium (YPD, 1% yeast extract, 2% peptone and 2% dextrose) or buffered complex glycerol medium (BMGY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 0.4 mg/mL biotin, 1% glycerol). P. pastoris was induced in buffered complex methanol medium (BMMY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 0.4 mg/mL biotin, 0.5% methanol). YPD plates containing 1 M Sorbitol and 2.5 mg/mL geneticin sulfate were used for selection of positive strains containing the pPIC3.5 K expression vector. E. coli was cultivated in LB medium (0.5% yeast extract, 1% peptone, 1% NaCl). F. graminearum was also grown in YPD medium.