Cloning and Purification of VviDREBA1 Proteins

The full VviDREBA1s [GenBank Accession No. MF445007 (VviDREBA1-1), MF445008 (VviDREBA1-6) and MF445009 (VviDREBA1-7)] open reading frames (ORFs), including stop codons, were amplified by RT-PCR using the primers included in the Supplementary Table S1. The forward VviDREBA1-7 and VviDREBA1-1 primers contained a BamHI site, and the forward VviDREBA1-6 primer contained a XhoI site. The three reverse primers contained an EcoRI site. The resulting fragments digested with their respective restriction enzymes were cloned into the pTrcHisA vector, which contains an N-terminal His₆ (Invitrogen, Carlsbad, CA, United States), previously digested with the same enzymes, and transformed into BL21-CodonPlus (DE3)-RIL competent cells. The induction and purification of recombinant proteins were performed according to Romero et al. (2008) (link). The purified fusion proteins were concentrated as described by Romero et al. (2016) (link). Protein analyses were performed on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) using Mini-Protean II Cell (Bio-Rad) equipment as described by Rosales et al. (2014) (link). Western blots were probed with antibodies and conditions previously described by Romero et al. (2016) (link).