Formation of active inclusion bodies in the periplasm of <i>Escherichia coli</i>

Arié, Jean‐Philippe; Miot, Marika; Sassoon, Nathalie; Betton, Jean‐Michel

doi:10.1111/j.1365-2958.2006.05394.x

Cited by 78 publications

(73 citation statements)

References 41 publications

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“…This differential centrifugation procedure was mandatory for the global analysis of periplasmic protein aggregates. It missed, however, a fraction of periplasmic protein aggregates that had already pelleted during the first centrifugation step (27). 4,5 For the analysis of MalE, OppA, HisJ, and MglB, samples were centrifuged for 10 min at 20,000 ϫ g immediately after the spheroplasting procedure, and supernatants containing the periplasmic soluble proteins were withdrawn.…”

Section: Methodsmentioning

confidence: 99%

Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB

Malki¹,

Le²,

Milles

et al. 2008

Journal of Biological Chemistry

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The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100 -5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1-bis(4-anilino)naphtalene-5,5-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

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Section: Methodsmentioning

confidence: 99%

Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB

Malki¹,

Le²,

Milles

et al. 2008

Journal of Biological Chemistry

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“…This is in agreement with recent findings of Betton and coworkers who showed that despite its poor folding efficiency in the periplasm, a fusion of MalE31-Bla retained some catalytic activity. 29 Our previous findings showed that MalE31 expressed in the cytoplasm is somewhat soluble, albeit to a lesser extent than wt MBP. 22 It has been shown that the periplasmic chaperone FkpA can decrease aggregation of MalE31, whereas another periplasmic chaperone SurA did not affect MalE31 aggregation.…”

Section: Cis-and Trans-acting Factors That Affect Protein Folding In mentioning

confidence: 99%

“…For these studies, we used E. coli MBP and a collection of MBP variants that become kinetically trapped in offpathway intermediates that are prone to aggregation. 29 One of these, a double mutant called MalE31 (G32D/I33P), reportedly forms inclusion bodies in the periplasmic space. 45 We cloned four different Fig.…”

Section: Cis-and Trans-acting Factors That Affect Protein Folding In mentioning

confidence: 99%

“…28 Hence, a screen or selection for monitoring protein folding in the bacterial periplasm would be a desirable accomplishment, but to date has been met with technical difficulties. 29 In this study, we developed an activity-independent selection strategy that reliably reports the ''folding robustness'' of POIs expressed in the periplasm. The term folding robustness is used here to denote both the chemical solubility related to correct folding of the POI and the avoidance of aggregation or degradation.…”

Section: Introductionmentioning

confidence: 99%

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A rapid protein folding assay for the bacterial periplasm

et al. 2010

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An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N-terminal signal recognition particle (SRP)-dependent signal peptide and a C-terminal selectable marker, TEM-1 b-lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo b-lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding-enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein.

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“…Only coexpression of multiple cytosolic chaperones (DnaK, DnaJ, GrpE, GroES, and GroEL) combined with expression at room temperature and lower concentrations of the inducer, IPTG (isopropyl beta-D-thiogalactoside), resulted in reasonable levels of soluble cytosolic expression and bioactivity comparable to native IL-6. 69 Even though there have been some successful attempts to produce disulfide-bonded proteins in E. coli using various strategies 70 other than the multiple chaperone approach required for IL-6, some of these attempts have unexpectedly resulted in the production of periplasmic inclusion bodies 71 and expression of proteins with nonnative disulfide bonds, 72 as was most likely the case for IL-6. 69 Thus, the limitations of E. coli for the expression of human proteins can be overcome but only by testing multiple expression conditions, including localization and coexpression of chaperones.…”

Section: Expression Hosts For Soluble Targetsmentioning

confidence: 99%

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

Cuozzo¹,

Soutter²

2014

SLAS Discovery

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Production of novel soluble and membrane-localized protein targets for functional and affinity-based screening has often been limited by the inability of traditional protein-expression systems to generate recombinant proteins that have properties similar to those of their endogenous counterparts. Such targets have often been labeled as challenging. Although biological validation of these challenging targets for specific disease areas may be strong, discovery of small-molecule modulators can be greatly delayed or completely halted due to target-expression issues. In this article, the limitations of traditional protein-expression systems will be discussed along with new systems designed to overcome these challenges. Recent work in this field has focused on two major areas for both soluble and membrane targets: construct-design strategies to improve expression levels and new hosts that can carry out the posttranslational modifications necessary for proper target folding and function. Another area of active research has been on the reconstitution of solubilized membrane targets for both structural analysis and screening. Finally, the potential impact of these new systems on the output of small-molecule screening campaigns will be discussed.

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Formation of active inclusion bodies in the periplasm of Escherichia coli

Cited by 78 publications

References 41 publications

Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB

Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB

A rapid protein folding assay for the bacterial periplasm

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

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