Quality control of overexpressed membrane proteins

Geertsma, Eric R.; Groeneveld, Maarten; Slotboom, Dirk-Jan; Poolman, Bert

doi:10.1073/pnas.0802190105

Cited by 168 publications

(207 citation statements)

References 23 publications

(32 reference statements)

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“…However, we were unable to demonstrate a significant difference in the level of SDS-resistant protein aggregates by SDS-PAGE between WT and RNAi-AtATG18a plants (data not shown). 57 While we cannot rule out an effect on aggregated protein that is not detectable by this procedure, these results indicate that autophagy may act in some as yet unidentified role during these stresses.…”

Section: Forward Primersmentioning

confidence: 77%

Autophagy is required for tolerance of drought and salt stress in plants

Liu¹,

Xiong²,

Bassham³

2009

Autophagy

362

340

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Autophagy is a protein degradation process in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon the induction of autophagy, a double membrane autophagosome forms around cytoplasmic components and delivers them to the vacuole or lysosome for degradation. In plants, autophagy has been shown previously to be induced during abiotic stresses including nutrient starvation and oxidative stress. In this paper, we demonstrate the induction of autophagy in high salt and osmotic stress conditions, concomitant with the upregulation of expression of an Arabidopsis thaliana autophagy-related gene AtATG18a. Autophagy-defective RNAi-AtATG18a plants are more sensitive to salt and drought conditions than wild-type plants, demonstrating a role for autophagy in the response to these stresses. NADPH oxidase inhibitors block autophagy induction upon nutrient starvation and salt stress, but not during osmotic stress, indicating that autophagy can be activated by NADPH oxidase-dependent or -independent pathways. Together our results indicate that diverse environmental stresses can induce autophagy and that autophagy is regulated by distinct signaling pathways in different conditions.

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Section: Forward Primersmentioning

confidence: 77%

Autophagy is required for tolerance of drought and salt stress in plants

Liu¹,

Xiong²,

Bassham³

2009

Autophagy

362

340

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“…This has been attributed primarily to the high hydrophobicity of membrane proteins, which complicates high-level production, purification, and crystallization (25). Consequently, yields are often frustratingly low, as underscored by a series of elegant screens for membrane protein overproduction in Escherichia coli (10,11,15,47). Moreover, the accumulation of overproduced proteins in biological membranes may affect bilayer integrity, which would be toxic for the producing cell (33).…”

mentioning

confidence: 99%

Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis

Zweers

Wiegert

Dijl

2009

Appl Environ Microbiol

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Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the W regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.

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“…By cloning the target gene into the NcoI-PstI sites, the gene is inserted into the correct frame and fused to the His-tag. This His-tag was obtained from expression vectors previously shown to perform efficiently when used in protein overproduction, immunodetection and purification settings [40,55].…”

Section: Discussionmentioning

confidence: 99%

An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis

et al. 2014

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Background: Scientific interest in Enterococcus faecalis has increased greatly over recent decades. Some strains are involved in food fermentation and offer health benefits, whereas others are vancomycin-resistant and cause infections that are difficult to treat. The limited availability of vectors able to express cloned genes efficiently in E. faecalis has hindered biotechnological studies on the bacterium's regulatory and pathogenicity-related genes. The agmatine deiminase (AGDI) pathway of E. faecalis, involved in the conversion of agmatine into putrescine, is driven by a response inducer gene aguR.Results: This study describes that the exposure to the induction factor (agmatine) results in the transcription of genes under the control of the aguB promoter, including the aguBDAC operon. A novel E. faecalis expression vector, named pAGEnt, combining the aguR inducer gene and the aguB promoter followed by a cloning site and a stop codon was constructed. pAGEnt was designed for the overexpression and purification of a protein fused to a 10-amino-acid His-tag at the C-terminus. The use of GFP as a reporter of gene expression in E. faecalis revealed that under induction with 60 mM agmatine, fluorescence reached 40 arbitrary units compared to 0 in uninduced cells. Conclusion: pAGEnt vector can be used for the overexpression of recombinant proteins under the induction of agmatine in E. faecalis, with a close correlation between agmatine concentration and fluorescence when GFP was used as reporter.

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Quality control of overexpressed membrane proteins

Cited by 168 publications

References 23 publications

Autophagy is required for tolerance of drought and salt stress in plants

Autophagy is required for tolerance of drought and salt stress in plants

Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis

An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis

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