Ni2 nta resin

All chemicals were of reagent grade and purchased from commercial suppliers including Aladdin (Shanghai, China), Yuanye Bio-Technology (Shanghai, China), Sigma-Aldrich (St. Louis, MO, USA), unless otherwise noted. Phusion High-Fidelity DNA polymerase and restriction enzymes were purchased from Thermo-Fisher Scientific (Pittsburgh, PA, USA). T4 DNA ligase was purchased from New England Biolabs (Ipswich, MA, USA). DNA extraction kit and gel extraction kit were purchased from Promega (Madison, WI, USA). Oligonucleotide primers were ordered from GeneWiz (Suzhou, China). Ni²⁺-NTA resin was purchased from Genscript (Nanjing, China). Amicao® Ultra-15 filtration devices (molecular cutoff 10–100 KDa) were purchased from Millipore (Billerica, MA, USA). BCA protein assay kit and precast-gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchase from Solar Bio (Beijing, China).

Glycine, nicotinamide adenine dinucleotide (NAD⁺, NADH), Tris (2-carboxyethyl) phosphine (TCEP), THF and AOX enzyme (Pichia pastoris, A2404) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The derivatization reagents dansyl chloride and CAT enzyme (10⁵ U/ml) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Dithiothreitol (DTT), β-mercaptoethanol (β-ME), pyridoxal 5’-phosphate monohydrate (PLP) and flavin adenine dinucleotide (FAD) were obtained from Aladdin (Shanghai, China). Escherichia coli DH5α and BL21 (DE3) were used for plasmid construction and the overexpression of recombinant proteins, respectively. Ni²⁺-NTA resin was purchased from Genscript (Nanjing, China). Amicon® Ultra-15 filtration devices (molecular size cut-off 10 KDa for H protein, 30 KDa for T protein and L protein and 100 KDa for P protein) were purchased from Millipore (Billerica, MA, USA). Mut Express II Fast Mutagenesis kit V2 was purchased from Vazyme (Jiangsu, China). BCA protein assay kit and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were purchased from SolarBio Co., Ltd. (Beijing, China). Luria-Bertani (LB) medium containing tryptone (10 g/L), yeast extract (5 g/L) and NaCl (10 g/L) were used for cloning and expression.