Journal:

Article Title: Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding ▿

doi: 10.1128/JVI.00772-08

Figure Lengend Snippet: (A) FLAG-tagged E2 proteins were expressed in CV-1 cell lines, and the level of each protein was determined by immunoblot analysis using an anti-FLAG M2 antibody. E2-TA, wild-type E2 protein; E2-N, E2 N terminus; DBD, DNA binding domain; E2-TR, E2 repressor protein. (B) E2 W360G is defective in dimerization. Nuclear extracts from CV-1 cells expressing FLAG-tagged wild-type BPV-1 E2 (WT) and W360G proteins were fractionated by Superdex 200 10/300 GL column chromatography. The fractions were analyzed by immunoblotting using anti-FLAG M2 antibody. Gel filtration standards (Bio-Rad) were run in parallel to independently assess the sizes of the complexes and are indicated in kilodaltons. (C) FLAG-tagged E2-FKBP fusion proteins were expressed in CV-1 cell lines, and the level of each protein in the presence (+) or absence (−) of 10 nM AP20187 was determined by immunoblot analysis using an anti-FLAG M2 antibody.

Article Snippet: For the FKBP fusion proteins and controls, DNA encoding the FKBP domain was amplified from pC 4 -F v 1 E (Ariad Pharmaceuticals) and fused to the 3′ end of the E2 gene region encoding residues 1 to 285, resulting in an E2 N-terminal domain and hinge region (NH)-FKBP fusion protein.

Techniques: Western Blot, Binding Assay, Expressing, Column Chromatography, Filtration

Journal:

Article Title: Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding ▿

doi: 10.1128/JVI.00772-08

Figure Lengend Snippet: (A) In vitro-translated E2 proteins were tested for their abilities to bind Brd4 in the presence of increasing amounts of AP20187. Aliquots (4 μl) of reticulocyte lysate (adjusted for the concentration of E2) were assayed for binding to 5 μl of Brd4 protein extract prebound to anti-FLAG immunobeads. Bound proteins were eluted and analyzed by SDS-PAGE. E2-TA, wild-type E2 protein. (B) The optimal amount of AP20187 required to enhance E2-FKBP E2 binding to Brd4 was determined as described in the legend to panel A. Bound E2-FKBP was quantitated using a Molecular Dynamics Typhoon imager. Levels of bound E2 are expressed as percentages relative to the input.

Article Snippet: For the FKBP fusion proteins and controls, DNA encoding the FKBP domain was amplified from pC 4 -F v 1 E (Ariad Pharmaceuticals) and fused to the 3′ end of the E2 gene region encoding residues 1 to 285, resulting in an E2 N-terminal domain and hinge region (NH)-FKBP fusion protein.

Techniques: In Vitro, Concentration Assay, Binding Assay, SDS Page

Journal:

Article Title: Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding ▿

doi: 10.1128/JVI.00772-08

Figure Lengend Snippet: (A) The E2 proteins indicated were expressed in CV-1 cells, and the localization of each E2 protein in a complex with Brd4 was detected by indirect immunofluorescence. The percentage of mitotic E2-expressing cells that had recruited Brd4 in speckles to the mitotic chromosomes is shown, along with the standard deviation of results derived from several experiments. The bars labeled +AP show results for cells treated with 10 nM AP20187. E2-TA, wild-type E2 protein; DBD, DNA binding domain; E2-TR, E2 repressor protein. (B) Representative indirect immunofluorescence images of E2-FKBP proteins (green), including proteins in complexes with Brd4 (red), on mitotic chromosomes in the presence or absence of 10 nM AP20187. Cellular chromosomes are stained with DAPI (blue). Mitotic cells are indicated by arrows.

Article Snippet: For the FKBP fusion proteins and controls, DNA encoding the FKBP domain was amplified from pC 4 -F v 1 E (Ariad Pharmaceuticals) and fused to the 3′ end of the E2 gene region encoding residues 1 to 285, resulting in an E2 N-terminal domain and hinge region (NH)-FKBP fusion protein.

Techniques: Immunofluorescence, Expressing, Standard Deviation, Derivative Assay, Labeling, Binding Assay, Staining

Journal: bioRxiv

Article Title: Biophysical characterization and analysis of a mesophilic chorismate mutase from B. pumilus

doi: 10.1101/2023.04.20.537678

Figure Lengend Snippet: Heavy atom distances between the substrate hydroxyl group on C 4 and amide protons of Cys75 (BpCM in redBsCM in cyan) throughout the course of the chorismate mutase reaction.

Article Snippet: The gene encoding the Bacillus pumilus AroH-type Chorismate mutase (uniprot A8FEK3, BpCM) with a dual C-terminal stop codon (TAATAA) was codon optimized for expression in E . coli , synthesized and subcloned into pET22b by GenScript Biotech (Netherlands) B.V.

Techniques:

Journal: Scientific Reports

Article Title: Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase

doi: 10.1038/s41598-019-49984-3

Figure Lengend Snippet: Three-dimensional structure of the citrate complex of PgDPP11. ( a ) Dimeric structure of PgDPP11. One subunit is colored in rainbow colors from the N-terminus (blue) to the C-terminus (red), and the other is colored gray. ( b ) A stereo diagram showing the PgDPP11 subunit. The catalytic domain is colored in blue to cyan and orange to red. The α-helical domain is colored in yellow to green. The catalytic triad “Asp227-His85-Ser655” is marked by an ellipsoid. The bound citrate (green) and potassium (purple) ions are shown in ball-and-stick and sphere models, respectively. ( c ) The mode of citrate ion binding in the S1 subsite of PgDPP11. Possible hydrogen bonds and salt bridges are shown as dashed lines. ( d ) Comparison of the present crystal structure with a dipeptide (Leu-Asp, blue) docking model of PgDPP11. The bound water molecules except for HOH240 were removed for clarity.

Article Snippet: A synthetic gene encoding full-length PgDPP11 (residues 1-720, UniProt accession number B2RID1), codon-optimized for expression in E . coli , was purchased from Genscript (NJ, USA).

Techniques: Binding Assay, Comparison

Journal: Scientific Reports

Article Title: Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase

doi: 10.1038/s41598-019-49984-3

Figure Lengend Snippet: Data collection statistics for PgDPP11.

Article Snippet: A synthetic gene encoding full-length PgDPP11 (residues 1-720, UniProt accession number B2RID1), codon-optimized for expression in E . coli , was purchased from Genscript (NJ, USA).

Techniques:

Journal: Scientific Reports

Article Title: Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase

doi: 10.1038/s41598-019-49984-3

Figure Lengend Snippet: Stereodiagrams showing weighted m |Fo|- D |Fc| omit maps of the bound ligand molecule in the S1 subsite of PgDPP11. The contour levels are 4.0 σ (cyan). ( a ) Citrate ion at a 1.50-Å resolution. ( b ) SH-5 at a 2.39-Å resolution.

Article Snippet: A synthetic gene encoding full-length PgDPP11 (residues 1-720, UniProt accession number B2RID1), codon-optimized for expression in E . coli , was purchased from Genscript (NJ, USA).

Techniques:

Journal: Scientific Reports

Article Title: Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase

doi: 10.1038/s41598-019-49984-3

Figure Lengend Snippet: Mode of SH-5 binding in the S1 subsite of PgDPP11. The in silico model is shown as a stick model with hydrogens. ( a ) A 3D pharmacophore model for the first-stage screening. Hydrogen bond donor (HBD) and acceptor (HBA) features are shown as cyan and magenta spheres, respectively. ( b ) A stereo diagram showing in silico docking model (light green) based on MM-GBSA scoring and present crystal structure (yellow) at a 2.39 Å resolution. Possible hydrogen bonds and salt bridges are shown as thick and thin dashed lines for the crystal structure and docking model, respectively.

Article Snippet: A synthetic gene encoding full-length PgDPP11 (residues 1-720, UniProt accession number B2RID1), codon-optimized for expression in E . coli , was purchased from Genscript (NJ, USA).

Techniques: Binding Assay, In Silico

Journal: Scientific Reports

Article Title: Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase

doi: 10.1038/s41598-019-49984-3

Figure Lengend Snippet: Inhibitory effects of SH-5 and NPPB against DPPs.

Article Snippet: A synthetic gene encoding full-length PgDPP11 (residues 1-720, UniProt accession number B2RID1), codon-optimized for expression in E . coli , was purchased from Genscript (NJ, USA).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase

doi: 10.1038/s41598-019-49984-3

Figure Lengend Snippet: Comparison of the residues in the S1 subsite of DPP7-type and DPP11-type S46 peptidases. Residues that form hydrogen bonds with the nitro group of SH-5, Thr650 and Asn670 in PgDPP11 (Fig. ) and corresponding residues conserved in other DPP11s are coloured red. Arg673, which is crucial for the strict Asp/Glu specificity of PgDPP11, is shown in blue. The arginine residue is also conserved in PeDPP11 (Arg670) but is replaced by a serine residue (green) in SmDPP11 (Ser667) and SpDPP11 (Ser684).

Article Snippet: A synthetic gene encoding full-length PgDPP11 (residues 1-720, UniProt accession number B2RID1), codon-optimized for expression in E . coli , was purchased from Genscript (NJ, USA).

Techniques: Comparison, Residue

Journal: Scientific Reports

Article Title: Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase

doi: 10.1038/s41598-019-49984-3

Figure Lengend Snippet: Refinement statistics for PgDPP11.

Article Snippet: A synthetic gene encoding full-length PgDPP11 (residues 1-720, UniProt accession number B2RID1), codon-optimized for expression in E . coli , was purchased from Genscript (NJ, USA).

Techniques:

Journal: Biomolecules

Article Title: Rational Design of Key Enzymes to Efficiently Synthesize Phycocyanobilin in Escherichia coli

doi: 10.3390/biom14030301

Figure Lengend Snippet: Assembly of enzymes and supplement of precursor to improve PCB synthesis. ADB1 and ADB2 are two zinc finger proteins that can specially bind to DNA scaffold1 and scaffold2. Values and error bars represent means and standard deviations of biological triplicates.

Article Snippet: The heterologous genes, including ho T from Thermosynechococcus elongatus BP-1, ho S from Synechocystis sp. PCC6803, ho N from Nostoc sp. PCC 7120, ho NM from Neisseria meningitidis , ho GM from Glycine max , ho H from Homo sapiens , ho B from Bos taurus , ho O from Oryctolagus cuniculus , ho R from Rattus norvegicus , ho GG from Gallus gallus , pcyA S from Synechocystis sp. PCC6803, pcyA T from Thermosynechococcus elongatus BP-1, pcyA N from Nostoc sp. PCC 7120, pcyA SU from Synechococcus sp., pcyA P from Prochlorococcus sp ., ADB1 and ADB2 encoding zinc finger proteins, iRFP and Alr1966g2C56A encoding infrared fluorescence proteins were codon-optimized and synthesized by GenScript Biotech Co., Ltd. (Nanjing, China).

Techniques: