Mutant METTL5 was generated using a QuikChange Site‐Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The recombinant plasmid was transformed into competent E. coli BL21 (DE3) (Bioteke, Beijing, China), and the positive monoclones were inoculated into LB‐Amp medium (Solarbio). The transformed bacteria were placed on a shaker (37°C, 100 rpm) for 3‐5 h until they reached an optical density (OD) value of 0.6‐0.8. Isopropyl β‐D‐thiogalactoside (IPTG; Solarbio) was added to induce protein expression, and the cultures were allowed to grow overnight at 16°C and 180 rpm. The next day, the bacteria were collected and lysed using an EmulsiFlex B‐15 high‐pressure homogenizer (Avestin, Ottawa, ON, Canada). Then, the lysate was centrifuged at 14000 rpm for 30 min at 4°C, and the supernatant was collected. The supernatant was mixed with Ni‐NTA resin (Qiagen Inc., Valencia, CA, USA) and incubated at 4°C for more than 40 min. After extensive washing with buffers containing 10 mmol/L, 20 mmol/L and 50 mmol/L imidazole (Solarbio), the protein was eluted in buffer containing 250 mmol/L imidazole. The protein was purified using an AKTA system (AKTA purifier, GE Healthcare, Northampton, MA, USA).
Imidazole
Manufactured by Solarbio
Sourced in China
Imidazole is a heterocyclic organic compound with the chemical formula C3H4N2. It is a colorless, crystalline solid that serves as a core functional group in various biomolecules and chemical compounds. Imidazole is commonly used as a buffer, chelating agent, and intermediate in organic synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Akta purifier, by GE Healthcare (1 mentions)
Isopropyl β d thiogalactoside iptg, by Solarbio (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Emulsiflex b15, by Avestin (1 mentions)
Amine coupling kit, by GE Healthcare (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Sonicator, by Ningbo Scientz Biotechnology (1 mentions)
Coomassie brilliant blue r 250, by Solarbio (1 mentions)
3 protocols using imidazole
1
Recombinant METTL5 Protein Expression and Purification
2
Compound Library Screening and Characterization
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Based on the preliminary screening results by network pharmacology analysis (Additional file 1 : Fig. S1, S2, S3, S4, S5), the compounds library was built, containing 83 compounds, which were obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China) with HPLC purity > 98% (Additional file 1 : Table S2). PBS, Tris, DMSO, and imidazole were acquired from Solarbio (Beijing, China) and other commercial suppliers. The sensor Chip (CM5), acetate (pH 4.0), PBS-P, and the amine coupling kit were purchased from GE Healthcare Life Sciences (Boston, USA).
3
Purification of Recombinant Proteins
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The harvested host cells were suspended in Tris-HCl buffer (100 mM, pH 8.0, plus 50 mM NaCl). The suspension was sonicated in a sonicator (Scientz, Zhejiang, China) using five cycles of 400 W for 4 min each. The host cell debris and supernatant were separated by centrifugation (10 000 × g, 60 min, 4 • C). The supernatant obtained was filtered (molecular weight cut off: 0.45 μm) by filter membrane, and loaded on a Ni 2+ -Nitrilotriacetic acid (NTA)-based affinity column. The target protein was eluted with Tris-HCl buffer (pH 8.0) containing imidazole (Solarbio, Beijing, China) at different concentrations of 0, 20, 40, 60, 80, 100 and 200 mM, respectively. (Solarbio, Beijing, China) . Furthermore, the purity of the eluted protein samples was tested using 12% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie Brilliant Blue R-250 (Solarbio, Beijing, China).
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