Transetta de3

The maltose binding protein (MBP) tagged TaMYC4 and glutathione S-transferase (GST) tagged TaJAZ1 proteins were expressed in Escherichia coli strain Transetta-DE3 (TransGen biotech, CD801) by induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 18 °C for 16 h. Cells were collected by centrifugation and then resuspended with the column buffer (for MBP-TaMYC4; 20 mM Tris-HCl, 0.2 M NaCl, 0.5 M EDTA, 1 mM PMSF, 1 mM DTT, 1 × cocktail). After sonication, samples were centrifuged at 4 °C for 30 min and the supernatant was used for further assays. Equal volumes of MBP and GST-TaJAZ1 or MBP-TaMYC4 and GST-TaJAZ1 were incubated with amylose resin beads, and analyzed by immunoblotting with anti-GST (Cat# CW0144, CWbiotech, Beijing, China) and anti-MBP (Cat# CW0288, 453CWbiotech, Beijing, China) antibodies.

For expression in E. coli, the ORFs of NES, GES1 and GES2 were amplified by PCR and subcloned directly into the pMAL-c2X expression vecter (New England Biolabs). Details of the primers are given in Supplementary Table 1. The recombinant plasmids pMAL-c2X::TwNES, pMAL-c2X::TwGES1 and pMAL-c2X::TwGES2 were separately transformed into the E. coli strain Transetta(DE3) (TransGen Biotech) for a fusion expression, using the original pMAL-c2X as negative control. Cultures (200 mL) were grown in LB medium containing 100 mg/L ampicillin until optical density of the culture at 600 nm reached 0.6 to 0.8 and then induced with 0.4 mM isopropyl 1-thio-β-D-galactopyranoside (Sigma, USA) at 16 °C for 20 h at 200 rpm. The cell pellets were harvested by centrifugation (3000 g, 20 min, 4 °C) and stored at −80 °C until used for affinity purification with Amylose Resin (New England Biolabs).

PTD is a short peptide (YGRKKRRQRRR) that mediates protein entry into cells.²⁴ (link) The PTD-ATF6 or PPM1H coding sequence was inserted into pET-28a+ plasmids. After transfer into E. coli strain Transetta (DE3) (Transgen Biotech, China), the recombinant proteins were induced by isopropyl β-D-thiogalactoside (IPTG), and the supernatant was subjected to Ni affinity chromatography after ultrasonic lysis of the bacterial solution (6×His-Tagged Protein Purification Kit – Soluble Protein, CWBIO). Then proteins were concentrated using an ultrafiltration centrifuge tube (Millipore) with a filter pore size of 30 kDa. The purified His-PPM1H was used to perform the in vitro phosphatase assay. PTD-ATF6 was applied to incubate cells at a certain concentration, and the culture medium was changed after 48 h.

Plasmids pEASY^®-Blunt (TransGen Biotech Co. Ltd., Beijing, China) and pET-28a (+) (Novagen, Madison, WI, USA) were used for gene amplification and heterologous expression, respectively. Integration plasmid pPIC3.5K (Invitrogen, Carlsbad, CA, USA) was functional in Pichia strain GS115.
E. coli strains Trans1-T1 and Transetta (DE3) were obtained from TransGen Co. Ltd. and were used as a bacterial host for recombinant plasmids amplification and enzymes expression, respectively. Pichia pastoris GS115 was used as a eukaryotic host for heterologous expression of OcGlcAE1~3. The detailed plasmids and strains used in this study are provided in Table S1.
UDP-d-GalA was from CarboSource Services (University of Georgia, Athens, GA, USA). UDP-d-GlcA, NAD⁺ and NADH were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). All other chemicals used in this study were of analytical grade.

The open reading frame (ORF) of LJ4CL was cloned into the expression vector pGEX-4T-1 and transformed into Transetta (DE3) chemically competent cells (Beijing TransGen Biotech Co., Ltd., Beijing, China), respectively. The vector pGEX-4T-1 (+) allows inframe cloning of PCR products resulting in a GST-tag attached at the N-terminal end of the recombinant protein. Expression of the recombinant protein was induced by adding isopropyl-β-d-1-thiogalactopyranoside (IPTG) and cells were harvested at 9 h. The activity of 4CL was analyzed according to Voo et al. [51 (link)]. The 1 mL reaction mixture contained 50 μL crude enzyme, 0.2 mM 4-coumarate, 0.8 mM ATP, 7.5 mM MgCl₂, and 38 M CoA in 100 mM Tris-HCl buffer (pH 7.5). One unit of 4CL was defined as the amount of enzyme that causes a decrease in A333 of 0.01 units min⁻¹. Protein concentration in the extracts was determined using the Lowry method [52 (link)].

S. thermocarboxydus 41291 was obtained from the Agricultural Culture Collection of China (Beijing, China). The E. coli strains Trans1-T1 and Transetta (DE3) were purchased from TransGen (Beijing, China). The expression vector pCold I was purchased from Takara (Beijing, China).

The plasmids of the positive clones from phage ELISA were isolated, and the VHH gene was digested with NcoI and NotI and ligated to a modified pET-25b vector that contained the 38-amino acid sequence of streptavidin binding protein (SBP) between the NotI and XhoI restriction sites (Additional file 1: Fig. S1) The resulting vector was transformed into competent E. coli Transetta-DE3 (Transgene, Beijing, China). Expression of the recombinant sdAb was induced by 1 mM IPTG, and then the proteins were purified by the Ni–NTA Agarose (Qiagen, Germany) under native conditions. The expression and purification of the sdAb was analyzed by SDS-PAGE electroporation and western blot with HRP-streptavidin (Solarbio Life Sciences, Beijing, China).