Ni nta affinity chromatography column

The GPEHT protein was further purified under denatured conditions using a nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography column (Qiagen, Hilden, Gemrany), according to the Qiagen protocol (Qiagen and Valencia, 2003). Endotoxins were removed from the GPEHT protein using ε-poly-L-lysine-agarose (Pierce High-Capacity Endotoxin Removal Spin Column, 0.5 mL, #88274; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The dialysis membrane was prepared via boiling in 1% acetic acid and EDTA (1 mM). Additionally, the boiling of the sample was repeated a second time in distilled water. The dialysis membrane was filled with purified protein and put in PBS solution for 24 h at 4 °C. The PBS solution was changed every 8 h. The samples were passed through a 0.2 µm filter to obtain sterile protein, and the concentration was determined using a Bradford assay.

Primers for NbPTP6 cloning were designed without signal peptide, as shown in Table 1. The amplified DNA fragment without signal peptide of NbPTP6 (693 bp) was inserted into pMD19-T vector for sequencing and subcloned into pET32a (+) plasmid. The recombinant plasmid was transformed into Escherichia coli strain BL21 (DE3) for prokaryotic expression. Cells were cultured and induced at 37 °C with 0.2 M IPTG for 4 h, then the recombinant proteins were purified using a Ni-NTA affinity chromatography column (Qiagen, Beijing, China). Mice were immunized with rNbPTP6 protein mixed with Freund’s adjuvant (1:1; Sigma-Aldrich, St. Louis, USA). After four immunizations at intervals of 7 days, anti-NbPTP6 serum was obtained and stored at − 20 °C.

PykF, cobB, and patZ genes were amplified from the genomic DNA of E. coli BW25113. The PCR product and pET28b plasmid were digested with restriction endonuclease EcoRI and HindIII (TaKaRa Bio, Japan), then ligated with T4 DNA Ligase (TaKaRa Bio) and transformed into E. coli BL21 The strains verified by sequencing were cultured in LB medium containing 50 μg/mL kanamycin at 37°C. When OD₆₀₀ reached 0.6, 0.5 mM isopropyl-d-thiogalactoside (IPTG; Sigma-Aldrich, USA) was added and cultured for 5 h to induce protein expression. The bacteria were centrifuged for 8,000 × g at 4°C and washed 3 times with 0.01 M PBS. The collected cells were crushed with a 0.01 M PBS suspension and cracked under high pressure. The supernatant was obtained by centrifugation for 30 min at 12,000 × g. Finally, the recombinant protein was purified by an Ni-NTA affinity chromatography column (Qiagen, Germany).

The putative lip54q gene was amplified using PCR, and the two primers used are listed as follows: lip54q-forward (5′-CGC GGATCC ATG CAA GTG GCA AAA CCG CAG GAT C-3′), and lip54q-reverse (5′-CCC AAGCTT GGG CCA TGC TTA TCT TCG AGA AAG-3′). The underlined sequences in the primers are two restriction sites (BamH I and Hind III) that were used to construct expression vectors. The PCR products were digested and ligated to the expression vector, pET32a, and digested with the same two restriction enzymes. The ligation products were electroporated into E. coli BL21(DE3). The transformants were spread on LB plates containing tributyrin and cultured overnight at 37°C. The recombinant bacteria expressing Lip54q produced transparent hydrolysis circles around the colonies. The recombinant bacteria were inoculated into LB medium and cultured at 37°C to an OD₆₀₀ of 0.8. IPTG with a final concentration of 0.2 mM was added to LB medium and induced at 30°C and 150 rpm for 18 h. The cells were centrifuged and washed with pure water 2 times and then resuspended in PBS buffer. The cells were lysed by ultrasonication, and the disrupted cells were centrifuged. The supernatant was a crude enzyme solution. This solution was purified by using an Ni-NTA affinity chromatography column (Qiagen, Germany) according to the product manual. The purified recombinant enzyme was stored at 4°C for further study.

Compounds DHAP, DHA, l-GAL, AP from potato, isopropyl-β-d-thiogalactopyranoside (IPTG), polyphosphate, ATP, l-fructose, l-tagatose, glycerol, glucose, and antibiotics were purchased from Sigma-Aldrich. All restriction enzymes and DNA ligase were purchased from Novagen (Darmstadt, Germany). Ni–NTA affinity chromatography column was purchased from QIAGEN. The yeast extract and tryptone were purchased from OXOID LID, and brain heart infusion (BHI) was purchased from Becton, Dickinson and Company. All bacterial strains and plasmids are listed in Table 4.

Asgard Thor-Rab, RB and TRAPPC3 gene sequences and the human Rab11B gene sequence were codon-optimized (Escherichia coli), synthesized (GenScript), and subcloned into the pSY5 vector including an N-terminal HRV 3 C protease cleavage site and 8-histidine tag. Thor-Rab was initially synthesized in a pEX-A2J2 vector (Eurofins) and subcloned in the pSY5 vector. Asgard proteins and human Rab11B were expressed as described^{25 (link)}. The cell pellets were extracted with binding buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 20 mM imidazole) supplemented with 0.01% TritonX-100 (Nacalai), protease inhibitor cocktail (EDTA-free, Calbiochem) and 2 μl of 10,000 u/μl benzonase (Merck). Cell lysis was performed using an ultrasonic cell disruptor (Branson) with 5 s of pulse, 30-40% duty for 5 min. Proteins were loaded on a Ni-NTA affinity chromatography column (Qiagen), and washed with five column volumes of binding buffer. The N-terminal His-tag was removed by cleavage with HRV-3C protease at 4 °C, overnight. The eluted proteins in the binding buffer were subjected to a size exclusion chromatography (Enrich SEC 70, Bio-Rad) in 20 mM HEPES, pH 7.5, 150 mM NaCl. Proteins were pooled and concentrated with 10 kDa MWCO centrifuge filters (Merck).