Ni nta protein purification kit

The tsiV3^cp and truncated C-terminal vgrG3^cp (VgrG3^cpC) were cloned into the expressing vector pET30a with a His tag and pGEX-6p with a GST tag, respectively. BL21(DE3) cells with recombinant proteins were grown to an OD₆₀₀ of ~0.8 and induced with addition of IPTG (final concentration, 100 μM) and culturing at 16°C overnight. Cell cultures were harvested by centrifugation and lysed by sonication. Whole-cell lysate was centrifuged at 4°C to remove precipitate, and then the supernatant was loaded on a Ni²⁺-nitrilotriacetic acid (NTA) column or GST Sefinose resin column to purify the proteins of interest. The GST recombinant protein purification kit (catalog number C600327-0001) and Ni-NTA protein purification kit (catalog number C600332-0001) were purchased from Sangon Biotech. All eluted proteins were further purified with the AKTA Explorer protein purification system.

The strain used was Sphingopyxis sp. USTB-05, capable of biodegrading MCs and NODs, previously isolated from Dianchi, China [48 (link)]. To construct a recombinant protein-expressing strain carrying the mlrB gene, we used the plasmid-amplified vector strain E. coli TOP10 and the expression strain E. coli BL21, which were purchased from Sangon Biotechnology Ltd. in Shanghai, China. Naturally, the cyclic vectors pET-30a having two histidine tags, both BamH I and Xho I restriction enzymes, the plasmid isolation kit, the polymerase chain reaction (PCR) kit, and the Ni-NTA protein purification kit were also all obtained from Sangon in Shanghai, China. The recombinant strains were grown in Luria-Bertani (LB) medium at 200 r/min at 37 °C. Ampicillin and Kanamycin were purchased from Biorigin (Beijing) Inc. (Beijing, China), which can select bacteria and prevent contamination during growth. Standard MC-LR (C₄₉H₇₄N₁₀O₁₂) of 99% purity was purchased from Shanghai Aibixin Biotechnology Co. Imidazole and standard MC-RR (C₄₉H₇₅N₁₃O₁₂, 95% purity) were purchased from Shanghai Malin Biochemistry Co., Shanghai, China. All other reagents used in the purification process were analytic grade.

Availability of adequate amounts of the pure allergens is essential to further understand their molecular structures, which is a pre-requisite for the development of more efficacious allergen immunotherapy. The amino acid sequences and coding genes of Hsp70 were retrieved from Uniprot database. The gene of Hsp70 was synthesized and cloned into prokaryotic expression vector pET-28a (+). The expression of His-tagged recombinant Hsp70 in transformant E. coli BL21 was induced by IPTG (final concentration = 1 mmol/L), and the expression products were analyzed by SDS-PAGE. Then, the bacteria were collected and disrupted by sonication. Hsp70 was purified by Ni-NTA protein purification kit (Sangon, C600320-0001), and the recombinant protein was eluted with elution buffer (containing 50mM Tris, 300mM NaCl and 500mM imidazole, Ph 8.0). Then, the endotoxin was removed by Endotoxin Removal Spin Columns (Thermo Fisher, NO. 88273). Purified protein was resuspended in 1×phosphate buffered solution (PBS), and analyzed by SDS-PAGE. The protein concentration was measured by the protein concentration assay kit (Sangon, C503071-0250).