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.
Highlights
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]
To synthesize the PldA-green fluorescent protein (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)
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
Summary
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]. 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 and expensive procedure with a low yield [2]. 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
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