*Article* **Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy**

**Svetlana I. Bakholdina 1,\*, Anna M. Stenkova 2, Evgenia P. Bystritskaya 1, Evgeniy V. Sidorin 1, Natalya Yu. Kim 1, Ekaterina S. Menchinskaya 1, Tatiana Yu. Gorpenchenko 3, Dmitry L. Aminin 1,4, Nikita A. Shved 2,5 and Tamara F. Solov'eva 1,\***


**Abstract:** The effect of cultivation temperatures (37, 26, and 18 ◦C) on the conformational quality of *Yersinia pseudotuberculosis* phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 ◦C, while at 37 ◦C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 ◦C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in *E. coli*, as well as for visualization of IBs interactions with eukaryotic cells.

**Keywords:** recombinant phospholipase A1; *Yersinia pseudotuberculosis*; inclusion bodies; fusion protein; green fluorescent protein
