Shuffle t7 express competent e coli

The expression vector of pET17b was purchased from Novagen (Madison, Wisconsin, USA). Cloning E. coli strain TOP10 was from Invitrogen (Carlsbad, Canada). Purified BSA100X (10 mg/mL), restriction endonucleases AgeI HF and SacI HF, the expression host of SHuffle T7 Express Competent E. coli and one of the PNGase F enzymes were purchased from New England Biolabs (Ipswich, Massachusetts, USA). CarboClip PNGase F was ordered from Asparia Glycomics (San Sebastián, Gipuzkoa, Spain). T4 DNA ligase and PageRuler Prestained Protein Ladder were from Thermo Scientific (Waltham, Massachusetts, USA). Cells were cultured in LB Broth medium and LB Agar (Scharlau, Barcelona, Spain). Isopropyl β-d-1-thiogalactopyranoside (IPTG) was from BioChemica (Billingham, UK). EDTA-free Mini Complete Protease Inhibitor was purchased from Hoffman-La Roche (Basel, Switzerland). Template DNA (pGEX-3x-PNGase F) containing the coding sequence for PNGase F was kindly provided by Professor Miklos Guttman (University of Washington, Seattle, Washington, USA). DNA sequencing was performed by Macrogen Europe (Amsterdam, the Netherlands). The HiTrap Chelating Ni-affinity column was from GE Healthcare (Chicago, Illinois, USA). The human IgG (hIgG1) sample and all other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

The LuHNL mutants were prepared via site-directed mutagenesis using the PrimeSTAR Mutagenesis Basal kit (Takara) with forward and reverse primers of a 27-mer oligonucleotide designed as indicated by the kit manual. The PCR was performed for 30 cycles: (denaturation 98 °C/10 s, annealing 55 °C/15 s, elongation 72 °C/40 s). The amplified PCR product was purified using a Wizard SV gel and PCR clean-up system (Promega). The resulting PCR product was transformed into JM109 E. coli competent cell. The recombinant plasmids were extracted from the JM109 E. coli and sequenced by Genetic Analyzer 3500 (ThermoFisher Scientific) using T7 promoter primer (5′-TAATACGACTCACTATAGGG-3′) and T7 terminator primer (5′-ATGCTAGTTATTGCTCAGCGG-3′). The confirmed plasmids were transformed into SHuffle T7 Express Competent E. coli (New England Biolabs) for expression. The purification of the mutants was performed as the protocol described for wild type.

Escherichia coli DH5α, kanamycin, restriction endonucleases (EcoRI and XhoI) and Isopropyl β-d-1-thiogalactopyranoside (IPTG) were purchased from Takara Co., Ltd. (Dalian, China). SHuffle T7 Express Competent E. coli was purchased from New England BioLabs (Beijing, China). pET28a expression vector was purchased from Stratagene (La Jolla, CA, USA). Ni²⁺-nitrilotriacetate (Ni²⁺-NTA) resin was obtained from Qiagen Inc., (Valencia, CA, USA). The hydrolytic substrate bis(p-nitrophenyl) phosphate sodium salt (BpNPP), l-α-glycerophosphorylcholine (GPC), l-α-lysophosphatidylcholine (LPC) and l-α-phosphatidylcholine (PC) used in the present study were all purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). All other chemicals used in the present study were of analytical grade.

The extracellular part of the IL-23 receptor (IL-23Rex, amino acids 24–350) coded by plasmid DNA (His₆–IL-23Rex–pET-28b [9 (link)] was produced in E. coli SHuffle strain (SHuffle T7 Express Competent E. coli, New England Biolabs, Ipswich, MA, USA). Bacterial cells were grown in LB broth with kanamycin (60 µg/mL) at 30 °C; protein production was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) after the culture reached the density OD₆₀₀ = 0.8, and the cells were cultivated overnight at 23 °C. The next day, the culture was collected by centrifugation and sonicated in TN buffer (50 mM Tris, 300 mM NaCl, pH = 8.0), after which the disrupted cells were spun down at 40,000 g for 20 min and supernatant was used for the purification of soluble protein using Ni-NTA agarose.