*2.7. Enzymatic Activity of Phospholipase A1 Associated with and Released from IBs*

As is known, IBs with a high protein content in the native or native-like conformation are easily soluble in the presence of relatively low molar concentrations of chaotropic agents [24,42]. The chimeric protein was solubilized from PldA-GFP IBs in 4 M urea. As shown above, the efficiency of IBs solubilization under these conditions was rather high, and the isolated recombinant protein contained a high percentage of β-structure. Parallel to this, IBs were dissolved under extremely harsh conditions in a traditional manner using high concentration of denaturant—8 M urea. The specific enzymatic activity of the PldA-GFP IBs suspended in a Tris-HCl buffer solution and the isolated recombinant protein solubilized in urea was determined after their incubation in the presence of Triton X-100 micelles, which is necessary for the formation of a functionally active spatial structure of PldA [35].

No studied samples of isolated PldA-GFP fusion protein and IBs exhibited enzymatic activity. The lack of phospholipase A1 activity is probably due to oligomerization of GFP, as it is known that the monomeric state of GFP is a necessary condition for maximum preservation of the native function of the target protein [43]. In addition, the properties of

the linker sequence (flexibility, length, hydrophobicity, etc.) could affect the conformation and activity of the fusion protein [22].

To confirm the GFP inhibitory effect on the fusion protein enzymatic activity, PldA was expressed in *E. coli* at 18 ◦C and PldA IBs were isolated and purified. The PldA IBs suspended in a Tris-HCl buffer solution showed an enzymatic activity of 5 μmol/min per 1 mg. The detected IBs enzymatic activity despite its low level demonstrates the fundamental possibility of obtaining functionally active PldA IBs. The PldA, which was solubilized from IBs with 4 M urea, had a 5-fold higher activity (25–30 μmol/min per 1 mg). Attention is drawn to the fact that the recombinant protein in the functionally active form was obtained by passing the stage of complete unfolding in 8 M urea. It should be noted that PldA IBs expressed at 37 ◦C were inactive [33].

#### *2.8. Interaction of PldA-GFP IBs with Neuroblastoma Cells*

The ability of PldA-GFP IBs to penetrate eukaryotic cells was studied. It was found that IBs of the chimeric protein is not toxic to neuronal cells in the range of concentrations from 0.4 to 100 μg/mL (Figure 10).

**Figure 10.** Cytotoxic activity of PldA-GFP IBs on mouse neuroblastoma Neuro-2a cells. The data are presented as the mean ± SEM values (*n* = 3).

However, it was found that IBs have the property to bind the neuroblastoma Neuro-2a cells and subsequently penetrate them (Figure 11A–C). According the analysis of electron microscopic images of Neuro-2a cells and PldA-GFP IBs, the size of the IBs of the chimeric protein is in the range of 800–1000 nm (Figure 11D,E).

According to fluorescence image analysis, the average amount of PldA-GFP IBs penetrating the neuroblastoma Neuro-2a cell in control (0 h) is not detectable (data not shown), in 1 h is 0.05 ± 0.03, in 4 h is 1.4 ± 0.5, while in 24 h this value increases almost 5 times to 5.5 ± 1.4 IBs particles per cell in comparison with 1 h (Figure 11F).

In summary, phospholipase A1 fused with GFP was expressed at different growth temperatures (37, 26, and 18 ◦C). The conformational quality of PldA-GFP IBs, as reflected by its specific fluorescence emission, enhanced by producing them at low expression temperatures. In this regard, the membrane beta-barrel protein phospholipase A1 is similar to most soluble globular proteins. The maximum content of the fusion protein in the nativelike conformation was found in IBs produced at 18 ◦C. We studied the size, stability in denaturant solutions, and enzymatic and biological activity of these IBs, and the secondary structure of the recombinant protein forming them.

**Figure 11.** Fluorescence image of mouse neuroblastoma Neuro-2a cells after incubation with PldA-GFP IBs (20 μg/mL) for 4 (**A**) and 24 h (**B**). Images are obtained by superimposing the passing and fluorescent channels. Fluorescence of IBs is shown in green (GFP), cell nuclei are shown in blue (Hoechst 33342). An enlarged image (×5) of a neuroblastoma cell with included PldA-GFP IBs inside (**C**); Scanning electron microscopy of Neuro-2a mouse neuroblastoma cells with adsorbed PldA-GFP IBs (**D**); and magnified image (×5) of PldA-GFP IBs alone (**E**). The average amount of PldA-GFP IBs in a single neuroblastoma Neuro-2a cell after incubation for 1, 4, and 24 h (**F**). The fluorescent images of 150 randomly selected cells for each time point were inspected, and the amount of fluorescent IBs penetrating the cell surface was calculated and expressed as number of PldA-GFP IBs per cell. The data are presented as the mean ± SEM values (*n* = 150); \* *p* < 0.05 compared with 1 h.

PldA-GFP IBs have fluorescent activity which indicates the presence of the recombinant protein in a native-like conformation. According to the CD spectroscopy data, isolated PldA-GFP has a high β-sheet content. Amyloid-like structures were also found in the studied IBs. Solubilization of the chimeric protein from IBs with urea and SDS is accompanied by a modification of its secondary structure. The nature and degree of structural changes in the protein induced by these denaturants depends on the nature of the denaturant, its concentration, and incubation time of IBs. It was found that intermolecular interactions holding properly folded PldA-GFP molecules in IBs may be less resistant to denaturant action than intramolecular ones, which provide the spatial structure of the protein. This suggests that the recombinant protein can be released from IBs while maintaining the native-like conformation when using mild conditions for its solubilization.

The presence of a fluorescent marker in a properly folded chimeric protein provided additional information on the molecular organization of IBs. According to the data obtained, PldA-GFP IBs are structurally heterogeneous: compactly packed, properly folded, proteolytic resistant and structurally less organized, more sensitive to the action of denaturants and proteinase K, protein molecules coexist in IBs. We also found differences in the structural organization of the fluorescently active polypeptides in IBs.

Comparative characteristics of PldA-GFP IBs and PldA IBs, produced in *E. coli* under the same conditions, showed that fusion with GFP enables more correct folding of PldA, but negatively affects its enzymatic activity. This fact is possibly due to the GFP type and the linker structure, used in these experiments. At the same time, IBs formed by PldA at 18 ◦C had enzymatic activity. It was firstly found that a membrane protein embedded in IBs showed functional activity. As can be assumed from the data obtained, PldA fused with a monomeric type of GFP will form enzymatically active IBs at low temperatures; this remains to be clarified in our further studies.

Chimeric protein IBs is not toxic to eukaryotic cells and has the property of penetrating into neuroblastoma cells.

In the course of the work, it was shown that the GFP marker can be useful not only as an indicator of the target protein folding, but also as a tool for studying the structural organization of IBs, their morphology and localization in *E. coli* cells, as well as for visualizing IBs interaction with eukaryotic cells.

The culture of neuroblastoma cells was selected for experiments as eukaryotic cells do not have the ability to phagocytosis and, therefore, do not absorb PldA IBs in this way. In addition, Neuro-2a neuroblastoma cells are brain tumor cells. Our data confirm the possibility of delivering functionally active proteins to tumor cells using IBs, including for antitumor therapy.
