*2.1. Effect of the Cultivation Temperature Downshift on the Expression of the PldA-GFP Fusion Protein*

To synthesize the PldA-GFP fusion, we have constructed the recombinant plasmid containing sequences of *pldA* encoding *Y. pseudotuberculosis* membrane bound phospholipase A1 and *gfp* encoding CopGFP based on pRSETa vector (Figure S1). The expression of the PldA-GFP at different cultivation temperatures (37, 26, and 18 ◦C) was induced by adding 0.1–1 mM IPTG followed by incubation for 3, 5 and 16 h. The amount of recombinant protein (based on intensity of the protein band on the SDS-PAGE) and the level of GFP fluorescence were determined for each experiment.

*E. coli* strain BL21(DE3) pLysS/PldA-GFP expressed the major protein in the region of 57 kDa corresponding to the calculated molecular weight of the expected PldA-GFP fusion protein. It was found that lowering the cultivation temperature from 37 to 26 ◦C significantly increased the recombinant protein production (Figure 1B). A further decrease in the growth temperature to 18 ◦C did not have a noticeable effect on the level of protein synthesis. The maximum GFP fluorescence was observed in *E. coli* grown at 18 ◦C, it decreased by almost four times during bacterial cultivation at 26 ◦C, and was close to the negative control—plasmidless cell level at 37 ◦C (Figure 1A). Such effects of cultivation temperature on the level of PldA-GFP expression and GFP fluorescence were observed at all studied time points (data not shown), while the IPTG concentration did not significantly change the level of recombinant protein synthesis and GFP fluorescence of the fusion protein (Figure 1).

Thus, the low cultivation temperature promoted an increase in GFP fluorescence in the chimeric protein and, consequently, an increase in the proportion of the correctly folded form of phospholipase A1.

Fluorescence confocal microscopy was used to study the morphology of the recombinant protein PldA-GFP and its localization in the cell. Figure 2 shows that homogeneous fluorescence throughout the cell cytoplasm was observed for bacteria expressing GFP only (Figure 3A), which is characteristic of the GFP soluble form. At the same time, under the similar culture conditions, the cells containing the pRSETa/PldA-GFP plasmid accumulated the chimeric protein in the form of aggregates (inclusion bodies), as evidenced by inhomogeneous fluorescence (Figure 2B). A similar morphology of inclusion bodies formed by chimeric proteins with GFP and other fluorophores, or mutant GFPs prone to IB formation, has been described in several works [3,18,19].

**Figure 1.** The level of GFP fluorescence in *E. coli* cells (**A**) and the expression of PldA-GFP fusion protein (**B**) in *E. coli* cell lysates after 16 h of incubation at different cultivation temperatures and IPTG concentrations. BL—plasmidless cells (negative control); GFP—cells expressing GFP protein (positive control). Fluorescence and bacterial mass for cell lysates were normalized according to OD600. The experiments were performed in three biological replicates. Results show the mean ± standard deviation (SD). The asterisk (\*) indicates a significant difference (*p*-value < 0.05) in the fluorescence level between indicated groups.

**Figure 2.** Fluorescence microscopy images of *E. coli* cells expressing GFP (**A**) and PldA-GFP (**B**).

**Figure 3.** SDS-PAG electrophoresis (**A**) and Western blotting (**B**). (**A**): Whole cell lysates of *E. coli* BL plasmidless (2) and *E. coli* expressing PldA-GFP (3) and PldA (4); purified PldA-GFP IBs: pellet (5) and supernatant (6); purified PldA IBs: pellet (7) and supernatant (8); GFP: supernatant (9) and pellet (10); molecular weight standard (1). (**B**): PldA-GFP IBs (1); PldA IBs (positive control) (2); molecular weight standard (3). Western blotting was performed using mouse polyclonal antiserum to recombinant PldA.
