Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

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Self-assembling thermostable chimeras as new platform for arsenic biosensing

Scientific Reports ◽

10.1038/s41598-021-82648-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rosanna Puopolo ◽

Ilaria Sorrentino ◽

Giovanni Gallo ◽

Alessandra Piscitelli ◽

Paola Giardina ◽

...

Keyword(s):

Inclusion Bodies ◽

Association Constant ◽

Immobilized Enzyme ◽

Thermus Thermophilus ◽

Chimeric Protein ◽

The Self ◽

Chimeric Proteins ◽

Self Assembling ◽

Langmuir Isotherm Model ◽

Activity Loss

AbstractThe correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (KAsIII) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol−1 for ArsC-Vmh2 and to 1200 (± 300) L mol−1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).

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The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum

Applied and Environmental Microbiology ◽

10.1128/aem.01446-08 ◽

2008 ◽

Vol 74 (23) ◽

pp. 7431-7433 ◽

Cited By ~ 20

Author(s):

Mónica Martínez-Alonso ◽

Nuria González-Montalbán ◽

Elena García-Fruitós ◽

Antonio Villaverde

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Recombinant Proteins ◽

Fluorescent Protein ◽

Native Structure ◽

Functional Quality ◽

Soluble Aggregates ◽

Recombinant Polypeptides ◽

Green Fluorescent

ABSTRACT We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure.

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Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

Journal of Virology ◽

10.1128/jvi.74.23.11339-11346.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11339-11346 ◽

Cited By ~ 58

Author(s):

Vitaly Boyko ◽

Jessica van der Laak ◽

Jacqueline Ferralli ◽

Elena Suslova ◽

Myoung-Ok Kwon ◽

...

Keyword(s):

Amino Acids ◽

Green Fluorescent Protein ◽

Tobacco Mosaic Virus ◽

Inclusion Bodies ◽

Fluorescent Protein ◽

Movement Protein ◽

Leading Edge ◽

Intercellular Transport ◽

C Terminus ◽

Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

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Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis

Microbiology ◽

10.1099/mic.0.26322-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2615-2626 ◽

Cited By ~ 21

Author(s):

Jeanette E. Bröms ◽

Anna-Lena Forslund ◽

Åke Forsberg ◽

Matthew S. Francis

Keyword(s):

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

Infection Process ◽

Regulatory Control ◽

Eukaryotic Cells ◽

Null Mutant ◽

Type Iii ◽

In Trans ◽

Key Residues ◽

Distinct Role

The homologous pcrGVHpopBD and lcrGVHyopBD translocase operons of Pseudomonas aeruginosa and pathogenic Yersinia spp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. In Yersinia, this operon is also required for yop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability of popB/yopB and popD/yopD was investigated. Secretion of PopB produced in trans in a ΔyopB null mutant of Yersinia was only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement the yopB translocation defect. The Yersinia ΔyopD null mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependent yop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functional Yersinia type III secretion.

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Motif trap: a rapid method to clone motifs that can target proteins to defined subcellular localisations

Journal of Cell Science ◽

10.1242/jcs.112.23.4207 ◽

1999 ◽

Vol 112 (23) ◽

pp. 4207-4211

Author(s):

L.A. Bejarano ◽

C. Gonzalez

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Eukaryotic Cells ◽

Subcellular Localisation ◽

Dna Fragments ◽

Rt Pcr ◽

Rapid Procedure ◽

Target Proteins ◽

Protein Motifs ◽

Green Fluorescent

We have developed a rapid procedure termed Motif Trap (MT) to identify protein motifs that are able to target proteins to a distinct subcellular localisation in eukaryotic cells. By expressing random DNA fragments fused to green fluorescent protein (GFP), individual cells with the GFP localisation of interest are readily isolated allowing for the expressed DNA fragments to be cloned by RT-PCR. These can then be used to identify the corresponding full-length cDNAs. Using MT, we have identified patterns of GFP localisation which correspond to every major organelle and compartment. We have shown that MT is useful to identify new sequences that determine subcellular localisation as well as known targeting motifs.

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Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice

Molecular and Cellular Biology ◽

10.1128/mcb.00426-18 ◽

2018 ◽

Vol 39 (1) ◽

Cited By ~ 10

Author(s):

Takuya Tomita ◽

Shoshiro Hirayama ◽

Yasuyuki Sakurai ◽

Yuki Ohte ◽

Hidehito Yoshihara ◽

...

Keyword(s):

Cell Function ◽

Fluorescent Protein ◽

Embryonic Fibroblasts ◽

Α Subunit ◽

Biochemical Alterations ◽

Cellular Processes ◽

Responsible Gene ◽

Intracellular Protein Degradation ◽

Green Fluorescent

ABSTRACT The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α′ subunit of casein kinase II (CK2α′) as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

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A Comprehensive Review of Spirit Drink Safety Standards and Regulations from an International Perspective

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-319 ◽

2017 ◽

Vol 80 (3) ◽

pp. 431-442 ◽

Cited By ~ 5

Author(s):

Xiao-Na Pang ◽

Zhao-Jie Li ◽

Jing-Yu Chen ◽

Li-Juan Gao ◽

Bei-Zhong Han

Keyword(s):

Maximum Level ◽

The United States ◽

International Perspective ◽

Control Measures ◽

Alcoholic Beverages ◽

Hydrocyanic Acid ◽

The European Union ◽

Safety Standards ◽

Standards And Regulations

ABSTRACT Standards and regulations related to spirit drinks have been established by different countries and international organizations to ensure the safety and quality of spirits. Here, we introduce the principles of food safety and quality standards for alcoholic beverages and then compare the key indicators used in the distinct standards of the Codex Alimentarius Commission, the European Union, the People's Republic of China, the United States, Canada, and Australia. We also discuss in detail the “maximum level” of the following main contaminants of spirit drinks: methanol, higher alcohols, ethyl carbamate, hydrocyanic acid, heavy metals, mycotoxins, phthalates, and aldehydes. Furthermore, the control measures used for potential hazards are introduced. Harmonization of the current requirements based on comprehensive scope analysis and the risk assessment approach will enhance both the trade and quality of distilled spirits. This review article provides valuable information that will enable producers, traders, governments, and researchers to increase their knowledge of spirit drink safety requirements, control measures, and research trends.

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Nuclear pores as versatile reference standards for quantitative superresolution microscopy

10.1101/582668 ◽

2019 ◽

Cited By ~ 7

Author(s):

Jervis Vermal Thevathasan ◽

Maurice Kahnwald ◽

Konstanty Cieśliński ◽

Philipp Hoess ◽

Sudheer Kumar Peneti ◽

...

Keyword(s):

Cell Lines ◽

Fluorescent Protein ◽

Reference Standards ◽

Nuclear Pore ◽

Biologically Relevant ◽

Superresolution Microscopy ◽

Absolute Measurements ◽

Quantitative Fluorescence

AbstractQuantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

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A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies

Microbial Cell Factories ◽

10.1186/1475-2859-13-68 ◽

2014 ◽

Vol 13 (1) ◽

Cited By ~ 7

Author(s):

Govindan Raghunathan ◽

Ganapathiraman Munussami ◽

Hyojin Moon ◽

Hyun-jong Paik ◽

Seong Soo A An ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Inclusion Bodies ◽

Fluorescent Protein ◽

Green Fluorescent ◽

Intracellular Inclusion

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Nuclear Dynamics in Arabidopsis thaliana

Molecular Biology of the Cell ◽

10.1091/mbc.11.8.2733 ◽

2000 ◽

Vol 11 (8) ◽

pp. 2733-2741 ◽

Cited By ~ 72

Author(s):

Eva Chytilova ◽

Jiri Macas ◽

Elwira Sliwinska ◽

Susanne M. Rafelski ◽

Georgina M. Lambert ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Laser Scanning ◽

Fluorescent Protein ◽

Higher Plants ◽

Chimeric Protein ◽

Time Lapse ◽

Confocal Laser ◽

Long Distance ◽

General Applicability ◽

Living Plant

The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to β-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm. This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.

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