The Effect of Conditions of the Expression of the Recombinant Outer Membrane Phospholipase А1 from Yersinia pseudotuberculosis on the Structure and Properties of Inclusion Bodies

Бахолдина, С. И.; Сидорин, Е. В.; Khomenko, V. A.; Isaeva, Marina; Kim, N. Yu.; Bystritskaya, Evgeniya; Pimenova, E. A.; Соловьева, Т. Ф.

doi:10.1134/s1068162018020061

Cited by 4 publications

(7 citation statements)

References 22 publications

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“…As discussed above, the target recombinant protein is the major component of bacterial IBs (Neubauer, 2006). Its relative abundance in IBs varies according to the amino acid sequence and it also depends on the conditions of bacterial culture and gene expression (Strandberg & Enfors, 1991, Baig, et al, 2014, Bakholdina, et al, 2018. Bacterial strains with mutations affecting protein folding and metabolism (e.g.…”

Section: Biophysical Nanoscale Properties Of Ibsmentioning

confidence: 99%

Bacterial inclusion bodies are industrially exploitable amyloids

Marco

Ferrer-Miralles

García‐Fruitós

et al. 2018

FEMS Microbiology Reviews

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Section: Biophysical Nanoscale Properties Of Ibsmentioning

confidence: 99%

Bacterial inclusion bodies are industrially exploitable amyloids

Marco

Ferrer-Miralles

García‐Fruitós

et al. 2018

FEMS Microbiology Reviews

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“…As is known, the bacterial outer membrane proteins with the β-pleated sheet secondary structure have a high propensity to form amyloid-like structures [ 37 ]. We have previously shown the presence of amyloids in PldA IBs formed in E. coli at 37 °C [ 33 ].…”

Section: Resultsmentioning

confidence: 99%

“…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 ].…”

Section: Resultsmentioning

confidence: 99%

“…PldA-GFP IBs and PldA IBs were isolated as described in previous work [ 33 ]. Briefly, wet bacterial biomass was lysed by lysozyme and exposed to ultrasound.…”

Section: Methodsmentioning

confidence: 99%

“…E. coli cells expressing GFP were lysed by lysozyme and exposed to ultrasound under the conditions described in previous work [ 33 ] and centrifugated at 4500× g for 30 min. GFP was isolated from supernatant by size-exclusion chromatography on a Superdex 200HR column in PBS with an elution rate of 0.5 mL/min using a FPLC system (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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.

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