Prokaryotic expression of CaNAC2c-GST and its purification
In order to obtain plenty of soluble CaNAC2c proteins, a pDEST-15
plasmid harboring T7:CaNAC2c-GST was transformed into theEscherichia coli (E.coli ) strain BL21 (DE3). Expression of
the fusion protein was induced with 1 mM IPTG (isopropyl
β-D-1-thiogalactopyranoside) at 20℃ for 12 h, and an SDS-PAGE assay was
performed to confirm whether the soluble fusion protein was present in
the supernatant of the E. coli strain BL21 (DE3) cell lysate. For
purification of the recombinant proteins with GST tag, the Beaver
BeadsTM GSH (Beaver Biosciences, China) were washed
three times by buffer A (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.4), and then mixed with
protein liquid slowly incubated for 3 h at 4℃. The beads were washed
five times by buffer A and the target proteins were eluted by buffer B
(50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0).