Selenium incorporation using recombinant techniques

Walden, Helen

doi:10.1107/s0907444909038207

Cited by 80 publications

(67 citation statements)

References 45 publications

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“…Buffers used for IMAC had 2 m m DTT added, while the desalting buffer had 5 m m reduced glutathione added to prevent loss of anomalous signal through oxidation 46.…”

Section: Experimental Methodsmentioning

confidence: 99%

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile

et al. 2017

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Clostridium difficile is a burden to healthcare systems around the world, causing tens of thousands of deaths annually. The S‐layer of the bacterium, a layer of protein found of the surface of cells, has received a significant amount of attention over the past two decades as a potential target to combat the growing threat presented by C. difficile infections. The S‐layer contains a wide range of proteins, each of which possesses three cell wall‐binding domains, while many also possess a “functional” region. Here, we present the high resolution structure of the functional region of one such protein, Cwp19 along with preliminary functional characterisation of the predicted glycoside hydrolase. Cwp19 has a TIM barrel fold and appears to possess a high degree of substrate selectivity. The protein also exhibits peptidoglycan hydrolase activity, an order of magnitude slower than that of lysozyme and is the first member of glycoside hydrolase‐like family 10 to be characterised. This research goes some way to understanding the role of Cwp19 in the S‐layer of C. difficile.DatabaseStructural data are available in the PDB under the accession numbers 5OQ2 and 5OQ3.

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“…Buffers used for IMAC had 2 m m DTT added, while the desalting buffer had 5 m m reduced glutathione added to prevent loss of anomalous signal through oxidation 46.…”

Section: Experimental Methodsmentioning

confidence: 99%

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile

et al. 2017

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“…Production of selenomethionine labelled proteins is possible in yeast with incorporation levels of around 50 % routinely reported for both P. pastoris and S. cerevisiae [20]. However higher selenomethionine incorporation has been reported.…”

Section: Yeast Cellsmentioning

confidence: 99%

“…The levels of incorporation for secreted glycoproteins is higher than for intracellular proteins as unlabelled protein is removed during the media exchange process [20]. For example, 85 % selenomethionine incorporation was achieved for envelope glycoprotein D from HSV1 [21] and 76 % for palmitoyl protein thioesterase 1 (PDB entry 1EI9 and 1EH5) [22].…”

Section: Insect Cellsmentioning

confidence: 99%

Structural Biology of Glycoproteins

Nettleship¹

2012

Glycosylation

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“…The expression and purification of Drep2 and FSP27 have been described in detail elsewhere (22,29). Drep4 CIDE was produced using a previously established method (30). Further details are provided in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

CIDE domains form functionally important higher-order assemblies for DNA fragmentation

Choi

Qiao

Hong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Cell death-inducing DFF45-like effector (CIDE) domains, initially identified in apoptotic nucleases, form a family with diverse functions ranging from cell death to lipid homeostasis. Here we show that the CIDE domains of Drosophila and human apoptotic nucleases Drep2, Drep4, and DFF40 all form head-to-tail helical filaments. Opposing positively and negatively charged interfaces mediate the helical structures, and mutations on these surfaces abolish nuclease activation for apoptotic DNA fragmentation. Conserved filamentous structures are observed in CIDE family members involved in lipid homeostasis, and mutations on the charged interfaces compromise lipid droplet fusion, suggesting that CIDE domains represent a scaffold for higher-order assembly in DNA fragmentation and other biological processes such as lipid homeostasis.CIDE family | higher-order structure | DNA fragmentation | apoptosis | lipid homeostasis A hallmark of apoptosis is the fragmentation of cellular genomic DNA into a laddered pattern composed of multiples of 180-to 200-bp pieces, which correspond to the DNA length in a nucleosome. Two decades ago, the enzyme responsible for regulated DNA fragmentation was identified from human HeLa cells as a heterodimeric complex of the nuclease DNA fragmentation factor (DFF) of 40 kDa (DFF40), and the inhibitor of 45 kDa (DFF45) (1). Independently, a heterodimeric complex of caspase-activated DNase (CAD) and its inhibitor (ICAD) was identified from mouse lymphoma cells (2, 3). DFF45 and ICAD chaperone the folding of DFF40 and CAD, respectively, and also trap them in inactive states through complex formation. On induction of apoptosis, DFF45 and ICAD are cleaved by activated caspases to release DFF40 and CAD for nuclear translocation and digestion of genomic DNA through large-scale chromatin fragmentation and internucleosomal DNA cleavage (1, 4).DNA fragmentation is the basis for the classical apoptotic TUNEL assay that detects DNA double-strand breaks (5). Because apoptotic cells display "eat-me" signals and are phagocytosed, DNA fragmentation has been proposed as a mechanism for avoiding the transformation of recipient cells by the activated oncogenes or viral genes and reduce the autoimmune response from the strong autoantigenic DNA (6). Clinically, sperm DNA fragmentation is used as a correlative to male infertility (7), and circulating fragmented cellfree DNA is detected as a disease biomarker (8).Human DFF40 and DFF45 and mouse CAD and ICAD contain a conserved N-terminal region known as the cell death-inducing DFF45-like effector (CIDE) domain (9) (Fig. S1A). In Drosophila, four DFF-related proteins (Drep1-4) are critical for apoptotic DNA fragmentation (10-12) (Fig. S1A), and Drep2 also acts as a unique synaptic protein important in learning and behavioral adaptation (13). Biochemical characterization of CIDE-CIDE interactions from Drep1 to Drep4 has revealed that the Drep2 and Drep4 nucleases interact with and are inhibited by . In addition to DNA fragmentation, the CIDE domain-containing proteins CIDEA...

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Selenium incorporation using recombinant techniques

Cited by 80 publications

References 45 publications

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile

Structural Biology of Glycoproteins

CIDE domains form functionally important higher-order assemblies for DNA fragmentation

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