E coli origami2 de3

Chitinase from S. griseus, HRP, cellulase from A. niger, N-acetyl-D-glucosamine and 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium salt were purchased from Sigma-Aldrich (St Louis, MO, USA). One unit of HRP is defined as the amount of enzyme that will form 1.0 mg of purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20°C. One unit of chitinase is defined as the amount of enzyme that liberates 1.0 mg of N-acetyl-D-glucosamine from chitin per hour at pH 6.0 at 25°C in a 2-hour assay. One unit of cellulase is defined as the amount of enzyme that liberates 1.0 μmol of glucose from cellulose in 1 hour at pH 5.0 at 37°C. 4-Aminoantipyrine was purchased from Acros Organics (Geel, Belgium), D-glucose monohydrate was purchased from Merck (Darmstadt, Germany) and cellotetraose, chitobiose and chitotetraose were purchased from Dextra (Reading, UK). Cellobiose (purity >98%) was purchased from TCI Europe (Zwijndrecht, Belgium). Whatman filter paper grade 1 was purchased from GE Healthcare Life Sciences (Little Chalfont, UK). E. coli ORIGAMI2 DE3 was purchased from EMD Millipore (Billerica, MA, USA) and pET-SUMO vector was obtained from Invitrogen (Carlsbad, CA, USA).

Total RNA from C. vulgaris was extracted from cells grown in high light using the Direct‐zol™ RNA Miniprep Plus kit (Zymo Research, Irvine, CA, USA). Transcript sequence was amplified from complementary DNA using specific primers designed on transcript g7391 (Supporting Information Methods S1). Mature VDE coding sequence was cloned into pET28 expression vector not including the initial 28 amino acids putative signal peptide for the chloroplast. The signal peptide was calculated using ChloroP 1.1 and TargetP 2.0 tools, considering the shortest result obtained. Potential additional residues involved in protein import in thylakoid lumen identified by TargetP are reported in Methods S1; these residues were anyway maintained in the recombinant C. vulgaris VDE protein produced, since they are not expected to influence protein activity. His‐tag was added at the C‐terminus. The pQE60 construct for expression of A. thaliana VDE enzyme in Escherichia coli, previously described (Hieber et al., 2002; Fufezan et al., 2012), was kindly gifted by Professor Tomas Morosinotto (University of Padua, Italy). Recombinant A. thaliana and C. vulgaris VDE were expressed in E. coli Origami™ 2(DE3) ( Merck KGaA, Darmstadt, Germany) by inducing cells with 1 mM isopropyl β‐d‐1‐thiogalactopyranoside for 5 h at 37°C and purified by nickel affinity column as described in Saga et al. (2010).