*2.2. Isolation and Purification of PldA-GFP IBs, PldA IBs, and GFP*

The proteins PldA-GFP, PldA, and GFP were expressed in *E. coli* at 18 ◦C, IPTG concentration of 0.1 mM, and cultivation time of 16 h (Figure 1). The bacterial cells were destroyed by lysozyme in combination with ultrasound, and the resulting suspensions were centrifuged. Using SDS-PAGE analysis, the recombinant proteins PldA-GFP and PldA were found only in insoluble fractions (pellets), while GFP was detected only in the supernatant (Figure 3A). These results confirm that in *E. coli* cells under mentioned expression conditions the chimeric protein and phospholipase A1 form IBs, while GFP is in a soluble form.

PldA-GFP IBs and PldA IBs were purified from impurities by sequential treatment with detergent solutions: 0.1% Sarcosyl and 1% sodium deoxycholate to remove LPS [20]. Denaturing SDS-PAGE showed that PldA-GFP and PldA migrate in the gel as bands corresponding to proteins with molecular weights of 57 and 31 kDa, respectively (Figure 3A). Both IBs samples barely contain any concomitant proteins. It should be noted that PldA-GFP loses GFP fluorescence under denaturing (2% SDS, boiled) and semi-native (0.1% SDS without boiled) SDS-PAGE. Immunoblotting using antibodies to PldA found the only protein band with MW of 57 kDa, which belongs to PldA-GFP (Figure 3B).

Recombinant GFP (rGFP) was isolated from the supernatant obtained by centrifugation of the lysed *E. coli* cells under the conditions described above using gel chromatography on a Superdex 200HR column. The fluorescent fractions were pooled and considered as containing GFP in its native conformation, as the formation of a fluorophore occurs spontaneously after this protein folding [21].

According to gel filtration data, GFP in PBS (pH 7.4) has an apparent molecular weight of approximately 42 kDa and, therefore, exists under these conditions as a dimer. Under denaturing (2% SDS and boiling) and semi-native SDS-PAGE (0.1% SDS without boiling), GFP migrates as a monomer with an apparent molecular weight of about 26 kDa, and loses fluorescence (Figure 3). Therefore, purified GFP is a dimer that has a fluorescence spectrum with maxima ex/em at 482/502 nm, and may be converted to a non-fluorescent monomer in the presence of a denaturant (SDS).

In phosphate-buffered saline PldA-GFP IBs have the same fluorescence spectrum as rGFP, and therefore contain the recombinant protein in a native-like conformation. The fluorescence intensity of IBs, calculated per mg of GFP, was four times lower than that of GFP (Figure 4). Therefore, in IBs only 24, 5%, of the chimeric protein is in a fluorescently active form. However, this value is not entirely correct. It can be either overestimated, as according to CD spectroscopy data, recombinant GFP contains a certain amount of an inactive protein; and underestimated, as is known, the aggregation of GFP leads to the loss of a large part of its fluorescent activity [22].

**Figure 4.** GFP fluorescence emission spectra of recombinant proteins in PBS: (1) GFP, (2) PldA-GFP IBs. The fluorescence intensity of GFP calculated per 1 mg of protein was taken as 100%.
