chaperone constructs  (New England Biolabs)

Journal: PLoS ONE

Article Title: A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

doi: 10.1371/journal.pone.0158923

Figure Lengend Snippet: Recombinant CHIKV nsP1 was purified with 6X his-mediated affinity chromatography followed by size exclusion chromatography. (A) Early attempts to purify CHIKV nsP1 resulted in highly aggregated protein that eluted exculsively in the void volume (eluted around 55 mL) during gel filtration, while protein purified after optimization of expression and purification conditions eluted around 72 mL. (B) Eluates resulting from size exclusion chromatography were concentrated and analyzed with SDS-PAGE gel electrophoresis and Coomassie staining. (C) This band was cut out, digested with trypsin and analysed further with orbitrap mass spectrometry analysis.

Article Snippet: The CHIKV nsP1 construct was transformed into Gro7 (Takara no. 3340) BL21 (DE3) E . coli competent cells (Novagen).

Techniques: Recombinant, Purification, Affinity Chromatography, Size-exclusion Chromatography, Filtration, Expressing, SDS Page, Nucleic Acid Electrophoresis, Staining, Mass Spectrometry

Journal: PLoS ONE

Article Title: A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

doi: 10.1371/journal.pone.0158923

Figure Lengend Snippet: Purified CHIKV nsP1 was incubated with GTP-680 and either 100 μM GTP, 100 μM SAM or 100 μM SAM and GTP for 1 hour. Reactions were resolved on SDS page gels and gels were scanned for fluorescence on an Odyssey Clx Infrared Imaging System before being stained with Commassie. (A) Guanylation was quantified and normalized to protein quantity using ImageJ and Image Studio software. Analysis of guanylation signal indicated a robust signal in Control and SAM only wells and depressed guanylation signals in GTP and GTP + SAM wells (gel shown is a representative gel). (B) Percent of control was calculated for Control, SAM, GTP and GTP + SAM wells. n = 3.

Article Snippet: The CHIKV nsP1 construct was transformed into Gro7 (Takara no. 3340) BL21 (DE3) E . coli competent cells (Novagen).

Techniques: Purification, Incubation, SDS Page, Fluorescence, Imaging, Staining, Software

Journal: PLoS ONE

Article Title: A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

doi: 10.1371/journal.pone.0158923

Figure Lengend Snippet: In order to determine the optimal pH for the FP assay, purified CHIKV nsP1 was incubated in the indicated buffer types with 10 nM GTP-Bodipy in black plates for 1 hour at 25°C in the dark. (A) Plates were then scanned for FP and signal windows were calculated for each buffer condition. (B) To determine the effect of DMSO concentration on FP assay signal window, purified CHIKV nsp1 was incubated with 10 nM GTP-Bodipy and various concentrations of DMSO ranging from 0.01%-10%. Plates were incubated for 1 hour at 25°C in the dark and then read for FP. (C) In order to determine the optimal assay volume, purified nsP1 was incubated with 10 nM GTP-Bodipy in FP binding buffer with 0.2% DMSO and plated in black plates in decreasing volumes from 65 μL to 10 μL. Plates were incubated for 1 hour at 25°C in the dark, scanned for FP and signal windows were calculated for each volume. (D) In order to determine the stability of the signal window over time, CHIKV nsP1 was incubated with 10 nM GTP-Bodipy at 25°C in the dark. Plates were scanned every 15 minutes for 120 minutes and signal windows were calculated at each time point. n = 3.

Article Snippet: The CHIKV nsP1 construct was transformed into Gro7 (Takara no. 3340) BL21 (DE3) E . coli competent cells (Novagen).

Techniques: FP Assay, Purification, Incubation, Concentration Assay, Binding Assay

Journal: PLoS ONE

Article Title: A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

doi: 10.1371/journal.pone.0158923

Figure Lengend Snippet: GTP and RNA cap analog competition analysis with CHIKV nsP1.

Article Snippet: The CHIKV nsP1 construct was transformed into Gro7 (Takara no. 3340) BL21 (DE3) E . coli competent cells (Novagen).

Techniques:

Journal: PLoS ONE

Article Title: A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

doi: 10.1371/journal.pone.0158923

Figure Lengend Snippet: nsP1 was incubated with GTP-680 and 100 μM of each GTP analog for 1 hour. Reactions were resolved on SDS-PAGE gels, scanned for fluorescence on an Odyssey Clx infared imager, and then stained with Coomassie. Guanylation was quantified and normalized to protein amount using ImageJ and Image Studio software. Percent of control (containing no GTP analog) was calculated for each inhibitor. n = 3.

Article Snippet: The CHIKV nsP1 construct was transformed into Gro7 (Takara no. 3340) BL21 (DE3) E . coli competent cells (Novagen).

Techniques: Incubation, SDS Page, Fluorescence, Staining, Software

Journal: PLoS ONE

Article Title: A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

doi: 10.1371/journal.pone.0158923

Figure Lengend Snippet: In order to determine if there were any drift or edge effects, we used an interleaved plate format and plated three types of FP signals in alternating columns. Minimum signal wells contained 10 nM GTP-Bodipy in FP binding buffer only, midpoint signal wells contained CHIKV nsP1, GTP-Bodipy in FP binding buffer, and 6.8 μM GTP and maximum signal wells contained CHIKV nsP1 and GTP-Bodipy in binding buffer. Plates were incubated for 1 hour at 25°C in the dark and scanned for FP. Values were plotted on a scatter plot with FP signal on the y-axis and (A) well by row, then column on the x-axis and also with (B) well by column, then row on the x-axis. n = 3.

Article Snippet: The CHIKV nsP1 construct was transformed into Gro7 (Takara no. 3340) BL21 (DE3) E . coli competent cells (Novagen).

Techniques: Binding Assay, Incubation