Bl21 de3 chemically competent cell

The vector construction and expression of recombinant SbIRP-1 were performed as previously described [37 (link)]. The coding region of SbIRP-1 was amplified with the primers SbIRP-Nco I and SbIRP-Xho I (Table 1). The amplified fragments were purified and digested with Nco I and Xho I before being inserted into the same double restriction enzyme linearized expression vector pET-28a. The constructed vector pET-28a-SbIRP-1 was transferred into BL21 (DE3) Chemically Competent Cell (TransGen, Beijing, China). Transformed cells were induced with 0.2 mM IPTG and cultured overnight at 18 °C. Histidine-labeled rSbIRP-1 was purified using ProteinPure Ni-NTA resin (TransGen) and concentrated by ultrafiltration after dialysis. For polyclonal antibody preparation, 2 mg of rSbIRP-1 protein was used as an antigen mixed thoroughly with an equal volume of Freund’s complete adjuvant and injected into New Zealand white rabbit at the first immunization, which was followed by a second and third immunization with 2 mg of rSbIRP-1 protein and the same volume of Freund’s incomplete adjuvant at 10-day intervals. The serum was harvested and stored at −80 °C before used. The specificity of the SbIRP-1 polyclonal antibody was tested by western blot of the rSbIRP-1 and hemocyte lysate.

Bovine serum albumin (BSA), human serum albumin (HSA), Brilliant blue R–250, Pyronin Y, DAPI and SYPRO (R) Orange Protein Gel Stain were purchased from Sigma Chemicals Co. All other chemicals are of analytical reagent grade. JM109, DH10B, BL21(DE3) Chemically Competent Cell, ProteinRuler I and ProteinRuler II, were purchased from TransGen Biotech (Beijing, China). A Pierce Rapid Gold BCA Protein Assay Kit was purchased from Thermo Fisher Scientific. RecR, RuvA, RuvB, RecA and MutL were cloned into an expression vector, and the proteins were purified. Cell cultures were maintained in the DMEM medium supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin storage solution (100 U/ml penicillin, 100 mg/ml streptomycin), at 37°C in a humidified atmosphere containing 5% CO₂. All the solutions were prepared with sterile ultrapure water. The (Z)-1,2-dimethyl-4-(pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (PyMDI) was synthesized according to the synthesis procedure described in [20 (link)].

The pEASY-T1 Cloning Vector plasmid containing positive clones and pET32a/pET28a plasmid were digested with BamHI and XhoI restriction enzymes for 3 h at 37 °C. Target fragments were purified and ligated into a digested pET32a plasmid. The recombinant plasmids were transformed into DH5α E. coli competent cells and grown on LB solid medium with 10 mL ampicillin (50 mg/mL). Then the BL21 (DE3) Chemically Competent Cell (TransGen, Wuhan, China) were transformed with correct recombinant plasmids. After a single clone was collected and cultivated overnight in LB liquid medium, the culture was added into fresh medium (1:100) and cultured at 37 °C for 4 h. Protein expression was induced by the addition of IPTG (isopropyl-beta D-thiogalactopyranoside, 0.5 mM). Cells were grown for 4 h at 37 °C, and then the culture were harvested by centrifugation (10,000 rpm, 10 min). Subsequently, the suspension was crushed by sonication and then separated into supernatant and sediment by centrifugation. Then, the Ni ion affinity chromatography (Thermo, USA) was used to purify target proteins from the supernatant. The His-tag of TintPBPs proteins were removed by enterokinase and their purity were analyzed by SDS-PAGE.