Hrv 3c protease

All reagents (analytical grade) and materials were purchased from commercial suppliers, such as nickel-NTA agarose (Qiagen, Valencia, CA, USA), human rhinovirus 3 C protease (HRV 3 C protease; TaKaRa, Otsu, Japan), inulin from dahlia tubers (Sigma–Aldrich, St. Louis, MO, USA), isopropyl-β-d-1-thiogalactopyranoside (IPTG; Amresco, Solon, OH, USA), silica gel G plate (Haiyang, Qingdao, China), and the expression vector pET-28a(+) (see Supplementary files) and strain Escherichia coli BL21 (DE3) (Synbio Technologies, Suzhou, China).
The exo-inulinase InuAGN25 and its encoding gene have been deposited in Genbank under accession numbers AGC01503 and JQ863108, respectively [3 ]. The gene was previously cloned from Sphingobacterium sp. GN25 and has been deposited in the China General Microbiological Culture Collection Center under CGMCC 1.10975 [3 ].

All reagents, purchased from commercial suppliers, were of analytical grade, including but not limited to human rhinovirus 3 C protease (HRV 3 C protease; TaKaRa, Otsu, Japan) and inulin from dahlia tubers (Sigma-Aldrich, St. Louis, MO, USA). The vector pET-28a(+) and strain E. coli BL21 (DE3) used for heterologous expression were supplied by Synbio Technologies (Suzhou, China).
Previously, Zhou et al. isolated Sphingobacterium sp. GN25 from feces of Grus nigricollis and deposited the strain in the China General Microbiological Culture Collection Center under CGMCC 1.10975 [10 ]. A novel low-temperature-active exo-inulinase, InuAGN25 (accession no. AGC01503), which can hydrolyze Jerusalem artichoke tuber powder solution to produce fructose at 0°C and 10°C, was identified from the strain based on the molecular activity strategy [10 ].

To purify the flWT-Kv3.1a tetramer, cells were resuspended in buffer A (Tris 20 mM pH 7.5, KCl 200 mM, MgCl2 1 mM, Lauryl Maltose Neopentyl Glycol 1%, cholesteryl hemisuccinate 0.1%, Protease inhibitor cocktail EDTA free from Roche according to manufacturer recommendations, and DNASE from Roche at 0.01 mg/ml). Cells were homogenized and the flWT-Kv3.1a tetramer was extracted by gentle stirring for 2 hours at 4°C. The nonsoluble fraction was removed by ultracentrifugation at 42,000 rpm at 4°C for 1 hour using Ti45 Beckman rotor. The soluble fraction was mixed with noncommercial Pro-GFP resin (anti-GFP binder coupled to CNBr Activated Sepharose resin from GE-healthcare) pre-equilibrated with buffer B (Tris 20 mM pH 7.5, KCl 200 mM, Lauryl Maltose Neopentyl Glycol 0.01%, and DTT 1 mM). After incubation for 2 hours at 4°C, the resin was washed extensively with buffer B and the flWT-Kv3.1a tetramer was eluted by addition of 100 µl of HRV 3C Protease at 1 U/µl concentration from Takara Bio. Elution was performed overnight at 4°C. Eluted protein was concentrated on vivaspin 100 kDa cutoff Vivaspin Turbo 15 concentrator and injected on a 10/30 Superose 6 size exclusion chromatography column pre-equilibrated with buffer B. Fractions, which correspond to the tetramer were pooled and concentrated to reach 1 mg/ml concentration.

To express the ALKBH6-His-taged fusion protein, the full-length cDNA encoding ALKBH6 was cloned into the pCold TF DNA vector (Takara, Shiga, Japan) using the BamHI/SalI sites. The resulting plasmid was transformed into the E. coli BL21 cell. After culturing the cells at 37 °C until OD₆₀₀ reached 0.4–0.8, the cultures were quickly cooled to 15 °C in ice water for 30 min, after which the synthesis of the recombinant protein was induced by adding 0.1 mM IPTG at 15 °C. The E. coli cells were collected, re-suspended in 1.0% PBS, pH 7.4, and disintegrated by sonication. The ALKBH6-His-taged fusion protein was digested with the HRV 3C protease (Takara), and the ALKBH6 protein was purified using a Ni-NTA His•Bind resin (Novagen, Temecula, CA, USA) using the elution buffer containing 250–500 mM imidazole. The total and purified proteins were analyzed via SDS-12% PAGE.

The expression host Pichia pastoris KM71H (Muts) (Invitrogen) was cultured on yeast extract-peptone-dextrose medium. BMGY (buffered glycerol-complex medium) at pH 6.5 was used for yeast growth and BMMY (buffered methanol-complex medium) at pH 6.5 was used for induction (Easy Select Pichia expression kit; Invitrogen). P. pastoris transformants were screened for protein induction in 24-well plates as described^{62 (link)}. Induction of protein expression was performed according to the manufacturer’s instructions. Purification of recombinant GFP, PsRLK6^ECD, and PsRLK7^ECD protein from the culture supernatant was performed by affinity chromatography using Ni-NTA Superflow resin. LRR5-6 region were recombinant expressed using E. coli strain BL21 (DE3) followed by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG, 0.5 mM; Sangon Biotech, A600168) at 18 °C for 16 h. Then purification of recombinant LRR5-6 TF protein from the culture supernatant was performed by affinity chromatography using Ni-NTA Superflow resin. Subsequently, HRV 3 C Protease (Takara, 7360) was employed to cleave the labeled LRR5-6 protein and produce unlabeled LRR5-6 protein.

The coding sequence of EF-1A was codon optimized and synthesized (General Biol Inc., China), and cloned into the pCold-II or pCold-TF vectors (TaKaRa Bio Inc., Japan). The pCold-II has a N-terminal His tag, whereas the pCold-TF contains a N-terminal His tag and a soluble trigger factor chaperone tag (TF) that contributes the solution of target protein. These resulting recombinant vectors were then transformed into Escherichia coli BL21 (TaKaRa Bio Inc., Japan) for protein expression. Briefly, the E. coli BL21 that bears the recombinant vector were inoculated in Luria-Bertani liquid medium containing 100 μg/mL, and incubated at 37 °C until the OD₆₀₀ reached 0.6, and were induced with 0.2 mM isopropyl β-D-1-thiogalactopyranodside at 15 °C for 16–18 h. The target EF-1A protein was purified by HisTrap HP column in ÄKATA Pure Protein Purification System (GE Healthcare Inc., USA). For protein that was expressed in pCold-TF vector, the TF tag was removed by HRV 3 C Protease (TaKaRa Bio Inc., Japan). All the purified EF-1A proteins were concerned by 10 K AIMCO Ultra-15 (Millipore Inc., USA), and were determined by Bradford Protein Assay Kit (Byotime Bio Inc., China).

E. coli TransT1 and E. coli BL21 (DE3) were obtained from TransGen (Beijing, China). DNA polymerase, DNA ladder, and protein markers were from Solarbio (Beijing, China). Exonuclease, HRV 3C Protease, and DNA ligase were from Takara (Dalian, China). Boc–Gln–Ala–Arg–AMC was from Bachem (Bubendorf, Switzerland). 6–isopropyl–β–d–thiogalactopyranoside (IPTG) was from Sigma–Aldrich (St. Louis, MO, USA). His antibody was from Beyotime (Shanghai, China). All other chemicals were of the highest reagent grade commercially available.