2.5 Heterologous protein expression and purification
Heterologous protein expression and purification were carried out following the methods of a previous study (Chen et al., 2020c) with slight modifications. The full-length sequence of CsGES was digested with Bam H1 and Sma l1, and the resulting gene fragments were cloned into the expression vector pGEX-4T-1 (Amersham Biosciences, Freiburg, Germany). The recombinant plasmids were transformed into E. coli strain BL21 (DE3) pLysS cells. The empty expression vector pGEX-4T-1 was transformed into E. coli BL21 (DE3) pLysS cells, which served as the negative control. After incubation at 37°C in Luria–Brentani liquid medium containing ampicillin (50 ug ml-1) and chloramphenicol (50 ug ml-1) for 20 to 24 h, the culture was diluted and grown until the optical density (OD600) of the cultured cells reached 0.6–0.8. Protein expression was induced by adding isopropyl-ß-D-thio-galactopyranoside to a final concentration of 1 mM. The cultures were incubated at 16°C with oscillation at 150 rpm for 22 h. Following IPTG induction, the cells were harvested at 4000 × g for 10 min; the expressed protein was then isolated and refolded as described in a previous study (Jing et al., 2019). The fusion proteins were purified by GST-binding resin (Novagen, Darmstadt, Germany) following the manufacturer’s protocol. The protein concentration was determined using a photometric method (Bradford, 1976) with bovine serum albumin as a standard. The correct size of the proteins was confirmed by SDS–PAGE.