Escherichia coli, BL, BL

Yang, Chengshuai, Li, Weichao, Li, Chen, Zhou, Zhihua, Xiao, Youli & Yan, Xing, 2018, Metabolism of ganoderic acids by a Ganoderma lucidum cytochrome P 450 and the 3 - keto sterol reductase ERG 27 from yeast, Phytochemistry 155, pp. 83-92 : 90

4.8. Heterologous expression of the ERG27 in E. coli BL21 (DE3)

The flanking sequences of ERG27 was PCR amplified with primers ERG27F and ERG27R using CEN.PK113-3C genomic DNA as the template. The fragment was linked into the plasmid pET28A (after Nco Ⅰ and Sal Ⅰ digestion) and transformed into E. coli BL21(DE3) using the ClonExpress Multis one Step Cloning Kit (Vazyme Biotech) to generate expression vector pET28A-ERG27 and recombined strain ZYERG27. The plasmid pET28A was transformed into E. coli BL21(DE3) as a control, named ZY28A. The induction and preparation of protein was carried out as described in our previous paper with slight modifications ( Yan et al., 2014). The control cells were treated in parallel. We used 100 mM Tris-HCl (pH 8.0) buffer for replacing 100 mM phosphate (pH 8.0) buffer. The in vitro enzymatic activity assays were conducted in 300 μL reactions of containing 100 mM Tris-HCl buffer (pH 8.0), 1 mM NADPH, 1 mM NADH, 50 mg /L substrate and 250 μL of crude enzymes. The reaction system was incubated at 30 ̊C for 12hr. The reactants were extracted with 300 μL n -butanol and analyzed by HPLC.