Proteinpure ni nta resin

Sequences encoding the BoTRAP2 truncated fragment of B. orientalis were cloned and ligated into the expression vector pET-28a. The expression vectors were transformed separately into E. coli BL21 (DE3) strain, and the soluble proteins were purified using ProteinPure Ni-NTA Resin (TransGen Biotech) according to the manufacturer’s instructions after the induction of 1 mM IPTG (Biosharp, Anhui, China) overnight at 28 °C.

The length of the BoSBP3-like gene is 3237 bp, which was too long for prokaryotic expression. The ORF of BoSBP3-like gene was truncated into three fragments: BoSBP3-like-1 (915 bp), BoSBP3-like-2 (1311 bp) and BoSBP3-like-3 (1011 bp), which were amplified and cloned into the expression vector pET-28a. The recombinant plasmids (pET-28a-BoSBP3-like-1, pET-28a-BoSBP3-like-2 and pET-28a-BoSBP3-like-3) were transformed separately into E. coli BL21 (DE3) strain. Small-scale culture was subjected to 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) induction overnight at 28 °C to identify the capacity and the forms of expression by SDS-PAGE analysis. BoSBP3-like-1/-2/-3 were expressed as His-fusion proteins, and purified using proteinPure Ni-NTA Resin (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Briefly, the transformed E. coli was washed three times with PBS (PH = 7.4), lysed by ultrasonication in binding buffer containing phenylmethylsulfonyl fluoride (PMSF), and then centrifuged at 15,000×g for 15 min at 4 °C. Supernatants containing the rBoSBP3-like-1/-2/-3 were purified with the Ni-NTA Resin (TransGen Biotech).

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.