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. 2022 Nov 17;13:1034230. doi: 10.3389/fpls.2022.1034230

Figure 1.

Figure 1

Preparation of recombinant MtCHIT5b protein and activity tests with Nod factor substrates. (A) Proteins eluted from nickel beads were separated on SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Ponceau staining of the membrane served as a loading control. Immunoblot analysis was performed with an anti-His antibody (B-G) Activity tests with obtained MtCHIT5b (0.25 μg mL-1) and Nod factors (60 μM) in a 100-μL test system. After incubation (37 °C) for 12 h, the Nod factors and acylated cleavage products were extracted with n-butanol and analyzed by reverse-phase HPLC. (B) Incubation of control proteins from E coli carrying the empty vector pET-28b with NodSm-IV(C16:2, S). (C) Incubation of MtCHIT5b with NodSm-IV(C16:2, S) resulting in II(C16:2) formation. (D) Incubation of empty vector control proteins with NodSm-V(C16:2, S). (E) Incubation of MtCHIT5b with NodSm-V(C16:2, S) resulting in II(C16:2) formation. (F) Incubation of empty vector control proteins with NodSm-IV(C16:2, Ac, S). (G) Incubation of MtCHIT5b with NodSm-IV(C16:2, Ac, S) resulting in II(C16:2, Ac) formation.