**1. Introduction**

The production of recombinant proteins in *Escherichia coli* is often accompanied by the formation of their insoluble aggregates, the so-called inclusion bodies (IBs), in the cell cytoplasm [1]. For a long time, it was believed that IBs consist of misfolded proteins, and the isolation of a recombinant protein from them in a functionally active form is a laborious

**Citation:** Bakholdina, S.I.; Stenkova, A.M.; Bystritskaya, E.P.; Sidorin, E.V.; Kim, N.Y.; Menchinskaya, E.S.; Gorpenchenko, T.Y.; Aminin, D.L.; Shved, N.A.; Solov'eva, T.F. Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy. *Molecules* **2021**, *26*, 3936. https://doi.org/10.3390/ molecules26133936

Academic Editor: Carmelo Corsaro

Received: 2 May 2021 Accepted: 22 June 2021 Published: 28 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and expensive procedure with a low yield [2]. However, it was later found that a protein in a native-like conformation can be present in IBs, and its amount can be controlled by manipulating the genetic and physiological parameters of expression [3]. A growing body of data confirms the fact that the protein within IBs adopts different conformational states ranging from unstructured, partly folded, native or native-like, to cross-β-sheet (amyloidlike) [4]. IBs containing a high percentage of properly folded protein can be functionally active and be used as nanomaterials in medicine and biotechnology [5]. The target protein from these IBs can be isolated under mild conditions without the use of renaturing steps and with high yield.

An important factor influencing the conformational quality of the protein in IBs is the expression temperature [6]. IBs synthesized at growth temperatures below the optimal 37 ◦C usually have a higher content of properly folded protein. The slower rate of protein production at low temperatures gives newly synthesized recombinant proteins more time to fold properly. Using low growth temperature, for a series of water-soluble globular proteins, including oligomeric ones, it was possible to obtain biologically active IBs with a high protein content in the native conformation, known as "nonclassical" IBs [7–9]. However, recently there was information that the globular protein L-asparaginase II forms "nonclassical" IBs, on the contrary, at elevated culture temperatures [10]. Moreover, it was reported that IBs formed at the optimal bacterial growth temperature contained a functionally active protein, and that the temperature regime did not affect the protein structure in IBs [11,12]. These facts demonstrate that the effect of the expression temperature on the conformational quality of IBs is ultimately determined by the nature of the protein, i.e., the intrinsic physicochemical properties of its amino acid sequence. At this time, the correlation between these factors affecting the structural organization of IBs is poorly understood. Most of the published research findings on this issue were performed on IBs of soluble globular proteins. IBs formed by membrane proteins are largely unexplored. In our work, the recombinant phospholipase A1 of *Yersinia pseudotuberculosis* as a model integral membrane protein was expressed in *E. coli* at different growth temperatures and the resulting IBs were characterized. In addition, determination of the growth temperature, which promotes the formation of IBs with a high content of correctly folded protein, can also be useful for developing novel, improved methods for biotechnology production of active enzyme. Lipolytic enzymes, including phospholipase A1, find wide application in food, cosmetic, and pharmaceutical industries [13].

The phospholipase A1 from the outer membrane protein of *Y. pseudotuberculosis*(PldA detergent-resistant phospholipase A1, EC 3.1.1.32) is a key enzyme that renews intracellular lipids (phospholipids) and thereby affects the permeability of the outer membrane, the functioning of various membrane-bound proteins, and is also one of the pathogenic factors of Gram-negative bacteria [14]. It has a molecular weight of 31 kDa. The PldA molecule is a β-barrel formed by a β-sheet, consisting of 12 antiparallel amphiphilic β-strands. In an active form, PldA is a homodimer [14].

IBs are of interest not only as a potential source of a functionally active recombinant protein, but also as a model system for studying protein aggregation associated with so-called conformational diseases, including numerous neurodegenerative disorders and amyloidosis [15]. Information about the mechanisms of formation and disaggregation of IBs, as well as their molecular organization, is important for understanding the molecular basis of conformational diseases, and in the design of drugs for inhibiting or reversing protein aggregation. However, such data on IBs are limited. This fact is largely determined by the methodological difficulties of studying these conformationally heterogeneous aggregates.

Green fluorescent protein (GFP) and GFP-like proteins were shown to be unique genetically encoded fluorescent markers for basic and applied research in the field of molecular and cellular biology. Currently, GFP are widely used to monitor the expression and folding quality of a recombinant protein under different bacterial culture conditions [6]. For this purpose, GFP is combined with the target protein so that the formation of a chromophore is possible only with the correct folding of the target protein [16,17]. This

approach significantly simplifies the process of searching for the expression conditions of a recombinant protein in the native conformation.

The aim of our work was to study the structure and properties of phospholipase A1 IBs formed at the optimal expression temperature in terms of their conformational quality, using a GFP tag as an indicator of recombinant protein folding. The research was focused on the following specific objectives: studying the effect of expression temperature on the conformational quality of PldA-GFP IBs; evaluating the secondary structure and arrangement of protein molecules inside the IBs; and collecting information about the stability and behavior of IBs in denaturing agents, as well as their enzymatic activity and interaction with eukaryotic cells.
