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

2009 ◽  
Vol 75 (23) ◽  
pp. 7356-7364 ◽  
Author(s):  
Jessica C. Zweers ◽  
Thomas Wiegert ◽  
Jan Maarten van Dijl
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Protein Production ◽  
Cell Envelope ◽  
Regulatory System ◽  
Interesting Candidate ◽  
High Level ◽  
And Function

ABSTRACT 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 σW 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.

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Nanoscale lipid membrane mimetics in spin-labeling and electron paramagnetic resonance spectroscopy studies of protein structure and function

2017 ◽  
Vol 6 (1) ◽  
pp. 75-92 ◽  
Author(s):  
Elka R. Georgieva
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Protein Structure ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Spin Labeling ◽  
Structure And Function ◽  
Protein Structure And Function ◽  
Paramagnetic Resonance ◽  
And Function ◽  
Electron Paramagnetic

AbstractCellular membranes and associated proteins play critical physiological roles in organisms from all life kingdoms. In many cases, malfunction of biological membranes triggered by changes in the lipid bilayer properties or membrane protein functional abnormalities lead to severe diseases. To understand in detail the processes that govern the life of cells and to control diseases, one of the major tasks in biological sciences is to learn how the membrane proteins function. To do so, a variety of biochemical and biophysical approaches have been used in molecular studies of membrane protein structure and function on the nanoscale. This review focuses on electron paramagnetic resonance with site-directed nitroxide spin-labeling (SDSL EPR), which is a rapidly expanding and powerful technique reporting on the local protein/spin-label dynamics and on large functionally important structural rearrangements. On the other hand, adequate to nanoscale study membrane mimetics have been developed and used in conjunction with SDSL EPR. Primarily, these mimetics include various liposomes, bicelles, and nanodiscs. This review provides a basic description of the EPR methods, continuous-wave and pulse, applied to spin-labeled proteins, and highlights several representative applications of EPR to liposome-, bicelle-, or nanodisc-reconstituted membrane proteins.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production

2016 ◽  
Vol 6 (2) ◽  
pp. 284-300 ◽  
Author(s):  
Dimitra Gialama ◽  
Kalliopi Kostelidou ◽  
Myrsini Michou ◽  
Dafni Chrysanthi Delivoria ◽  
Fragiskos N. Kolisis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Protein Production ◽  
High Level

Database Study on the Expression and Purification of Membrane Proteins

2021 ◽  
Vol 28 ◽  
Author(s):  
Chen-Yan china Zhang ◽  
Shi-Qi Zhao ◽  
Shi-Long Zhang ◽  
Li-Heng Luo ◽  
Ding-Chang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Drug Targets ◽  
Expression System ◽  
Data Bank ◽  
Hek293 Cells ◽  
Expression And Purification ◽  
Beta Barrel ◽  
Membrane Protein Expression

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

scholarly journals Plasma Membrane Protein Nce102 Modulates Morphology and Function of the Yeast Vacuole

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1476
Author(s):  
Katarina Vaskovicova ◽  
Petra Vesela ◽  
Jakub Zahumensky ◽  
Dagmar Folkova ◽  
Maria Balazova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Vacuolar Membrane ◽  
Yeast Culture ◽  
Membrane Domains ◽  
Biological Functions ◽  
Plasma Membrane Protein ◽  
Cell Architecture ◽  
And Function

Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.

scholarly journals A Novel Class of Heat and Secretion Stress-Responsive Genes Is Controlled by the Autoregulated CssRS Two-Component System of Bacillus subtilis

2002 ◽  
Vol 184 (20) ◽  
pp. 5661-5671 ◽  
Author(s):  
Elise Darmon ◽  
David Noone ◽  
Anne Masson ◽  
Sierd Bron ◽  
Oscar P. Kuipers ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Heat Stress ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Regulatory Region ◽  
Regulatory System ◽  
Secretion Stress ◽  
Two Component ◽  
High Level ◽  
Inducible Genes

ABSTRACT Bacteria need dedicated systems that allow appropriate adaptation to the perpetual changes in their environments. In Bacillus subtilis, two HtrA-like proteases, HtrA and HtrB, play critical roles in the cellular response to secretion and heat stresses. Transcription of these genes is induced by the high-level production of a secreted protein or by a temperature upshift. The CssR-CssS two-component regulatory system plays an essential role in this transcriptional activation. Transcription of the cssRS operon is autoregulated and can be induced by secretion stress, by the absence of either HtrA or HtrB, and by heat stress in a HtrA null mutant strain. Two start sites are used for cssRS transcription, only one of which is responsive to heat and secretion stress. The divergently transcribed htrB and cssRS genes share a regulatory region through which their secretion and heat stress-induced expression is linked. This study shows that CssRS-regulated genes represent a novel class of heat-inducible genes, which is referred to as class V and currently includes two genes: htrA and htrB.

scholarly journals Mutations of the YidC Insertase alleviate stress from σM-dependent membrane protein overproduction in Bacillus subtilis

2019 ◽  
Author(s):  
Heng Zhao ◽  
Ankita J. Sachla ◽  
John D. Helmann
Keyword(s):  
Oxidative Stress ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Substrate Binding ◽  
Single Amino Acid ◽  
Cell Wall Synthesis ◽  
Intrinsic Resistance ◽  
Σ Factor

AbstractIn Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in spoIIIJ, which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 SpoIIIJ contains a single amino acid substitution in the substrate-binding channel (Q140K), and this allele suppresses the lethality of high SigM. Analysis of a library of YidC variants reveals that increased charge (+2 or +3) in the substrate-binding channel can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidCQ140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.Author SummaryBacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis. In Bacillus subtilis, the alternative σ factor σM is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis. However, dysregulation of the σM regulon, resulting from loss of the YhdL anti-σM protein, is lethal. We here identify charge variants of the SpoIIIJ(YidC) membrane protein insertase that suppress the lethal effects of high σM activity. Further analyses reveal that induction of the σM regulon leads to high level expression of membrane proteins that trigger envelope stress, and this stress is countered by specific genes in the σM regulon.

scholarly journals Membrane-active Polymers: NCMNP13-x, NCMNP21-x and NCMNP21b-x for Membrane Protein Structural Biology

2022 ◽  
Author(s):  
Thi Kim Hoang Trinh ◽  
Claudio Catalano ◽  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Maleic Acid ◽  
Drug Targets ◽  
Design Synthesis ◽  
Native Cell ◽  
Wide Range ◽  
Lipid Particles ◽  
High Resolution Structure

Membrane proteins are a ubiquitous group of bio-macromolecules responsible for many crucial biological processes and serve as drug targets for a wide range of modern drugs. Detergent-free technologies such as styrene-maleic acid lipid particles (SMALP), diisobutylene-maleic acid lipid particles (DIBMALP), and native cell membrane nanoparticles (NCMN) systems have recently emerged as revolutionary alternatives to the traditional detergent-based approaches for membrane protein research. NCMN systems aim to create a membrane-active polymer library suitable for high-resolution structure determination. Herein, we report our design, synthesis, characterization and comparative application analyses of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x and NCMNP21b-x. Although each NCMN polymer can solubilize various model membrane proteins and conserve native lipids into NCMN particles, only the NCMNP21b-x series reveals lipid-protein particles with good buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. Consequently, the NCMNP21b-x polymers that bring out high-quality NCMN particles are particularly attractive for membrane protein structural biology.

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