E coli bl21 de3 plyss

The strains and plasmids used in this study were listed in additional file (Additional file 1: Table S1). E. coli Trans5α (TransGen, Beijing, China) was used for plasmid construction. E. coli BL21(DE3) and E. coli BL21(DE3)pLysS (TransGen, Beijing, China) were used as the hosts for Bgl1A(A24S/F297Y) production. Ampicillin, chloramphenicol, and IPTG were purchased from Sangon Biotech (Shanghai, China). p-Nitrophenyl β-D-glucopyranoside (pNPG) was from Sigma-Aldrich (St. Louis, MO, USA). The insoluble microcrystalline cellulose with an average particle size of 25 μm was acquired from Aladdin Chemistry (Shanghai, China). Ni²⁺-charged chelating sepharose fast flow was purchased from GE Healthcare (Uppsala, Sweden). All other chemicals were of analytical grade unless otherwise specified. Standard TB medium (per liter contains 4 g glycerol, 24 g yeast extract, 12 g peptone, 17 mM KH₂PO₄, and 72 mM K₂HPO₄) was used as culture medium in 250-mL Erlenmeyer flasks. Modified TB medium (per liter contains 15 g glycerol) was employed in high cell density culture (HCDC), the feeding solutions contained 45 g tryptone, 45 g yeast extract, and 500 g glycerol per liter.

DNA sequences encoding the cytoplasmic domain of BAK1, mBAK1 (L317E), SERK5 and SlBIR3 were amplified by PCR and subcloned into the pFLAG-MAC vector (Sigma-Aldrich Saint Louis, MO, USA). The primers used are listed in Table S1. Each of the resulting constructs was transformed into E. coli BL21(DE3)pLysS (Transgene, Beijing, China). The resulting recombinant proteins were purified as previously described and the autophosphorylation and their transphosphorylation activities in vitro were determined as previously described [77 (link)], using anti-FLAG (1:5000) (Transgene, Beijing, China, Cat. #HT201-01) and anti-pThr (1:2000) (CST, Danvers, MA, USA, Cat. #93815) antisera.