*3.4. Expression*

For protein expression, pRSETa/pldA-GFP, pRSETa/mPldA, and pRSETa/GFP constructs were transformed into *E. coli* BL21(DE3)pLysS competent cells. Single colonies were picked and inoculated in 10 mL of LB broth containing carbenicillin (100 mg/mL) and chloramphenicol (35 mg/mL) and grown overnight at 37 ◦C with shaking at 200 rpm. After 12 h, 250 mL of LB broth containing the same antibiotics was inoculated with 2% (*v*/*v*) of overnight grown primary culture and the culture was kept in incubator set at 37 ◦C with constant shaking at 200 rpm. IPTG induction was conducted at different concentrations (from 0.1 to 1 mM) when culture OD600 reached 0.5–0.7. The culture was further maintained at different temperatures, 37, 26, and 18 ◦C, for 3, 5, and 16 h. Before and after induction, approximately equal numbers of cells from various cultures (normalized according to OD600 values) were lysed in sample buffer and analyzed by SDS–PAGE. The GFP fluorescence of the chimeric protein PldA-GFP in *E.coli* cells was measured on the FL-600 Fluorescence/Absorbance Plate Reader (Bio TEK Instruments, Winooski, VT, USA). Absorption measurements (OD600) and fluorescence intensity measurements (λexc. 488 nm/λexp. 530 nm) were made on 100 μLcell suspension. Plasmidless *E. coli* BL21(DE3) strain was used as negative control, and *E.coli* BL21(DE3) with pRSETa/GFP as positive control under all expression conditions. The *E. coli* BL21(DE3)pLysS/mPldA strain was expressed at 18 ◦C for 16 h.

All measurements were performed in three biological replicates. Results were expressed as the mean ± standard deviation (SD). The Student's *t*-test was used to evaluate the data. Statistical significance was considered for *p*-values < 0.05.
