In vivo detection and quantification of chemicals that enhance protein stability

Hailu, Tsinatkeab T.; Foit, Linda; Bardwell, James C.A.

doi:10.1016/j.ab.2012.11.022

Cited by 18 publications

(14 citation statements)

References 32 publications

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“…The difficulty in reproducing the complex environment of living cells has led to the development of methods that allow the measurement of protein stability and folding kinetics inside the cell (8)(9)(10). Our lab recently developed biosensors that link protein stability to antimicrobial resistance, allowing the investigation of the periplasmic folding environment (11,12). Here, we describe the establishment of new protein folding sensors based on proteins involved with resistance to spectinomycin, kanamycin, and nourseothricin (ClonNAT) that expand our approach beyond the periplasm into the more complex folding environment of the cytosol.…”

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confidence: 99%

Cytosolic Selection Systems To Study Protein Stability

2014

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Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within guest proteins that affect their stability alter the antibiotic resistances of the cells expressing the biosensors in a manner that is related to the in vitro stabilities of the mutant guest proteins. In addition, we find that polyglutamine tracts of increasing length are associated with an increased tendency to form amyloids in vivo and, in our sandwich fusion system, with decreased resistance to aminoglycoside antibiotics. We demonstrate that our approach allows the in vivo analysis of protein stability in the cytosolic compartment without the need for prior structural and functional knowledge.

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confidence: 99%

Cytosolic Selection Systems To Study Protein Stability

2014

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“…These assays exploit β-lactamase's stability, the presence of permissive grafting sites in its structure, and the potential for high throughput screening via colorimetric assays. Here, we have shown the ability of the versatile tripartite β-lactamase platform 32,34,49 to distinguish aggregation-prone variants of Supplementary Fig. 13 shows an expanded view of residues 111-112.…”

Section: Discussionmentioning

confidence: 89%

An in vivo platform to select and evolve aggregation-resistant proteins

et al. 2020

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Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.

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“…To confirm in vivo PDI activity for DsbA2, we developed an assay in which the PDI detector TEM ␤-lactamase serves as the substrate. In this assay, gain of Bla activity, as measured with the chromogenic substrate nitrocefin, is de-pendent on PDI enzyme reducing and shuffling disulfide bonds until correct nonconsecutive disulfides are formed (29). As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Disulfide Bond Oxidoreductase DsbA2 of Legionella pneumophila Exhibits Protein Disulfide Isomerase Activity

et al. 2013

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The extracytoplasmic assembly of the Dot/Icm type IVb secretion system (T4SS) of Legionella pneumophila is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential disulfide bond oxidoreductase DsbA2 and not by DsbA1, a second nonessential DSB oxidoreductase. DsbA2, which is widely distributed in the microbial world, is phylogenetically distinct from the canonical DsbA oxidase and the DsbC protein disulfide isomerase (PDI)/reductase of Escherichia coli. Here we show that the extended N-terminal amino acid sequence of DsbA2 (relative to DsbA proteins) contains a highly conserved 27-amino-acid dimerization domain enabling the protein to form a homodimer. Complementation tests with E. coli mutants established that L. pneumophila dsbA1, but not the dsbA2 strain, restored motility to a dsbA mutant. In a protein-folding PDI detector assay, the dsbA2 strain, but not the dsbA1 strain, complemented a dsbC mutant of E. coli. Deletion of the dimerization domain sequences from DsbA2 produced the monomer (DsbA2N), which no longer exhibited PDI activity but complemented the E. coli dsbA mutant. PDI activity was demonstrated in vitro for DsbA2 but not DsbA1 in a nitrocefin-based mutant TEM β-lactamase folding assay. In an insulin reduction assay, DsbA2N activity was intermediate between those of DsbA2 and DsbA1. In L. pneumophila, DsbA2 was maintained as a mixture of thiol and disulfide forms, while in E. coli, DsbA2 was present as the reduced thiol. Our studies suggest that DsbA2 is a naturally occurring bifunctional disulfide bond oxidoreductase that may be uniquely suited to the majority of intracellular bacterial pathogens expressing T4SSs as well as in many slow-growing soil and aquatic bacteria.

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In vivo detection and quantification of chemicals that enhance protein stability

Cited by 18 publications

References 32 publications

Cytosolic Selection Systems To Study Protein Stability

Cytosolic Selection Systems To Study Protein Stability

An in vivo platform to select and evolve aggregation-resistant proteins

Disulfide Bond Oxidoreductase DsbA2 of Legionella pneumophila Exhibits Protein Disulfide Isomerase Activity

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