Deciphering correct strategies for multiprotein complex assembly by co-expression: Application to complexes as large as the histone octamer

Diebold, Marie-Laure; Fribourg, Sébastien; Koch, Michael; Metzger, Thibaud; Romier, Christophe

doi:10.1016/j.jsb.2011.02.001

Cited by 115 publications

(113 citation statements)

References 36 publications

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“…The cDNA encoding the CCT domain of , with the addition of a 59 ATG, was obtained by PCR amplification; the cDNA encoding CO CCT amino acids 290 to 352 with the R340Q mutation (Robson et al, 2001), At-NF-YA2 (amino acids 134-207), and At-NF-YA6 (amino acids 170-237) was obtained by gene synthesis (Eurofins Genomics) and cloned into pmcnEA/tH (Diebold et al, 2011) by restriction-end ligation to obtain C-terminal 6His-tag fusions . At-NF-YB2 mutant cDNA, encoding for amino acids 24 to 116 with residue Glu-65 mutated to Arg was obtained by gene synthesis and subcloned in pET15b to obtain the N-terminal 6His-tag fusion .…”

Section: Methods Protein Production and Purificationmentioning

confidence: 99%

“…At-NF-YB2 mutant cDNA, encoding for amino acids 24 to 116 with residue Glu-65 mutated to Arg was obtained by gene synthesis and subcloned in pET15b to obtain the N-terminal 6His-tag fusion . At-NF-YC9 cDNA, encoding amino acids 62 to 158 with a 59 ATG, a 39 stop codon, and mutant At-NF-YC9 with residue Phe-151 mutated to Arg (NF-YC9F151R) were obtained by gene synthesis and cloned in pmcnYC (Diebold et al, 2011). All constructs were verified by sequencing.…”

Section: Methods Protein Production and Purificationmentioning

confidence: 99%

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CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer

et al. 2017

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Section: Methods Protein Production and Purificationmentioning

confidence: 99%

Section: Methods Protein Production and Purificationmentioning

confidence: 99%

CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer

et al. 2017

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“…24 Overexpression was carried out in Escherichia coli BL21(DE3) cells containing the pRARE vector (Novagen, Darmstadt, Germany) in lysogeny broth medium. Induction was done at 25 °C by adding isopropyl-1-thio-β-D-galactopyranoside (IPTG; Euromedex, Souffelweyersheim, France) in a final assay concentration of 0.5 mM, in the presence of 100 µM ZnCl 2 .…”

Section: Proteinsmentioning

confidence: 99%

Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

et al. 2015

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Sirtuins are NAD + -dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC 50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.

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“…Within the SPINE2-COMPLEXES consortium a wide panel of cloning strategies and vector sets have been developed to streamline construct design for expression/co-expression screening in Escherichia coli (Busso et al, 2005;de Jong et al, 2006;Berrow et al, 2007;Scheich et al, 2007;Fogg and Wilkinson, 2008;Bieniossek et al, 2009;Unger et al, 2010;Diebold et al, 2011) (Luna-Vargas et al, 2011 as well as in insect and mammalian cells (Aricescu et al, 2006;Berrow et al, 2007;Abdulrahman et al, 2009;Pradeau-Aubreton et al, 2010;Trowitzsch et al, 2010). A variety of new technologies for DNA manipulation including ligation independent or restriction free procedures, in-fusion or gateway approaches are now being used in addition to classical restriction-based strategies (see Busso et al, 2011 for examples and test cases).…”

Section: Challenges For Sample Preparation: New Methods For Protein Cmentioning

confidence: 99%

Structural insights into transcription complexes

Berger

Blanco

Boelens

et al. 2011

Journal of Structural Biology

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a b s t r a c tControl of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of structural proteomics on our understanding of the molecular basis of gene expression. While most atomic structures were obtained by X-ray crystallography, the impact of solution NMR and cryo-electron microscopy is far from being negligible. Here, we summarize some highlights and illustrate the importance of specific technologies on the structural biology of protein-protein or protein/DNA transcription complexes: structure/ function analysis of components the eukaryotic basal and activated transcription machinery with focus on the TFIID and TFIIH multi-subunit complexes as well as transcription regulators such as members of the nuclear hormone receptor families. We also discuss molecular aspects of promoter recognition and epigenetic control of gene expression.

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Deciphering correct strategies for multiprotein complex assembly by co-expression: Application to complexes as large as the histone octamer

Cited by 115 publications

References 36 publications

CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer

CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer

Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

Structural insights into transcription complexes

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