Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin

Marchenko, Natalia; Sikorskaya, E.V.; Marchenkov, Victor V.; Kashparov, I. A.; Semisotnov, Gennady V.

doi:10.1016/j.pep.2015.11.020

Cited by 5 publications

(6 citation statements)

References 28 publications

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“…To verify the ability of GroEL 14 -EGFP to bind to GroES and denatured proteins, we used nondenaturing PAGE and affinity chromatography based on denatured lysozyme (see [ 35 ]). Figure 5 a presents the nondenaturing PAGE of the coexpression product GroEL 14 -EGFP without and with GroES and Mg-ADP and shows protein electrophoretic bands detected by Coomassie staining and fluorescence (see Materials and Methods ( Section 4 )).…”

Section: Resultsmentioning

confidence: 99%

“…The structural reconstruction of GroEL 14 and GroEL 14 -EGFP using relevant electron microscopy images supports this conclusion. GroEL 14 -EGFP was tested for the ability to interact with the cochaperonin GroES and a denatured protein, in our case, lysozyme lacking disulfide bonds [ 35 ]. The test confirmed that the GroEL 14 -EGFP particle retains its main functional features—the interaction with GroES and the denatured protein.…”

Section: Discussionmentioning

confidence: 99%

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In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties

et al. 2023

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The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties

et al. 2023

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“… GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work ( http://dx.doi.org/10.1016/j.pep.2015.11.020 [1] ), and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin) and denatured (lysozyme, α-lactalbumin and pepsin) proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.…”

mentioning

confidence: 80%

“…To choose the proteins for making affinity sorbents [1] , we evaluated the apparent dissociation constants of GroEL complexes with a number of proteins using titration experiments ( Fig. 1 , Fig.…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Dataset concerning GroEL chaperonin interaction with proteins

et al. 2016

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GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020[1]), and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin) and denatured (lysozyme, α-lactalbumin and pepsin) proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

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“…Chaperones have also been overexpressed in E. coli, purified, and immobilized on a chromatography column to refold apparently irreversibly denatured proteins, to renature insoluble proteins from an inclusion body, or to recondition enzymes that have lost their activity because of long storage [59]. GroEL is the most commonly used chaperone for in vitro refolding [59][60][61]. GroES in combination with GroEL, or with folding catalysts such as protein disulfide isomerase and peptidyl-prolyl isomerase, also proved to be beneficial [59].…”

Section: Environmental Applicationsmentioning

confidence: 99%

Potential Applications of the Escherichia coli Heat Shock Response in Synthetic Biology

Rodrigues

2018

Trends in Biotechnology

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The Escherichia coli heat shock response (HSR) is a complex mechanism triggered by heat shock and by a variety of other growth-impairing stresses. We explore here the potential use of the E. coli HSR mechanism in synthetic biology approaches. Several components of the regulatory mechanism (such as heat shock promoters, proteins, and RNA thermosensors) can be extremely valuable in the creation of a toolbox of well-characterized biological parts to construct biosensors or microbial cell factories with applications in the environment, industry, or healthcare. In the future, these systems can be used for instance to detect a pollutant in water, to regulate and optimize the production of a compound with industrial relevance, or to administer a therapeutic agent in vivo.

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Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin

Cited by 5 publications

References 28 publications

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties

Dataset concerning GroEL chaperonin interaction with proteins

Potential Applications of the Escherichia coli Heat Shock Response in Synthetic Biology

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