Escherichia coli, (Migula, 1895) Castellani & Chalmers, 1919, (Migula, 1895) Castellani & Chalmers

Zhang, Xinhua, Teixeira da Silva, Jaime A., Niu, Meiyun, Zhang, Ting, Liu, Huanfang, Zheng, Feng, Yuan, Yunfei, Li, Yuan, Fang, Lin, Zeng, Songjun & Ma, Guohua, 2021, Functional characterization of an Indian sandalwood (Santalum album L.) dual-localized bifunctional nerolidol / linalool synthase gene involved in stress response, Phytochemistry (112610) 183, pp. 1-12 : 10

5.5. Expression of recombination SaNES/LIS in E. coli and purification

For heterologous expression, the SaNES/LIS cDNA fragments were amplified using high-fidelity DNA polymerase PrimerStar (Takara Bio Inc.) and cloned into the pET-28a vector at Bam HI and Xho I sites using specific primers ( Table 2 ). The infusion constructs were verified by DNA sequencing at BGI. The recombinant plasmids were then transformed into the E. coli Rossetta 2 (DE3) strain. A single positive colony was incubated overnight at 37 ◦ C and shaken at 200 rpm in Luria-Bertani (LB) liquid medium with 34 μg/ml chloramphenicol and 100 μg/ml kanamycin. Overnight cultures were diluted 1: 100 with LB supplemented with the same antibiotics as described above until an OD 600 between 0.6 and 0.8 was achieved. IPTG was then added to a final concentration of 0.2 mM and the culture was incubated for an additional 24 h at 16 ◦ C and 150 rpm. Cells were harvested by centrifugation at 7000 g and 4 ◦ C for 5 min, and pellets were resuspended in lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4, pH 8.0) containing 1 mM PMSF and 0.5 mg /ml lysozyme. The incubated pellets were disrupted by sonication for 30 cycles (pulse on for 5 s, pulse off for 5 s, 30% amplitude) and the lysate was centrifuged at 12,000 rpm for 30 min. Recombinant His-tagged protein was purified by loading the lysate onto Ni-NTA Hispur Resin according to the manufacturer’ s protocol. The eluted proteins were further desalted on a PD-10 desalting column following the method by Jones et al. (2011). Protein concentration was determined by the Bradford (1976) method. SDS-polyacrylamide gel electrophoresis and Coomassie blue staining was used to confirm the presence of purified recombinant proteins.