Journal: Science Advances

Article Title: Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

doi: 10.1126/sciadv.add2191

Figure Lengend Snippet: ( A ) Plots of observed percent deuterium per residue for nsp7, nsp8, nsp9, nsp7-8, nsp7-10, and nsp7-11. Secondary structures from PDB 6YHU for nsp7 and nsp8, PDB 6WXD for nsp9, and PDB 6ZPE for nsp10 are drawn on plots with α helices shown as barrels, β strands shown as arrows, and coils shown as rectangles. ( B ) Scheme of integrative structural modeling workflow for the nsp7-11 polyprotein. One model is shown to represent all 10 generated models. ( C ) Top 10 nsp7-11 models grouped into four representative tertiary structures. Models are colored by nsp, with nsp7 in magenta, nsp8 in purple, nsp9 in teal, nsp10 in cyan, and nsp11 in tan.

Article Snippet: The pGEX-6P-1-nsp5 (or Mpro) plasmid was a gift from M. Walsh, Diamond Light Source. pGBWm4046979 (coding for full-length nsp7, National Center for Biotechnology Information (NCBI) reference sequence: YP_009725303.1, codon-optimized, with an initial Met and a cleavable C-terminal by tobacco etch virus (TEV) protease 6x-His tag] was a gift from Ginkgo Bioworks (Addgene plasmid 145611; http://n2t.net/ ; addgene:145611; RRID: Addgene_145611). pGBWm4046852 (coding for full-length nsp8, NCBI reference sequence: YP_009725304.1, codon-optimized, with an initial Met and a cleavable C-terminal TEV protease 6x-His tag) was a gift from Ginkgo Bioworks (Addgene plasmid 145584; http://n2t.net/ ; addgene:145584; RRID: Addgene_145584).

Techniques: Integrative Structural Biology, Generated

Journal: ACS Omega

Article Title: Production of Proteins of the SARS-CoV-2 Proteome for Drug Discovery

doi: 10.1021/acsomega.1c02984

Figure Lengend Snippet: Purification of the N-terminal ARH and PL pro domains of NSP3, NSP15, and NSP16 as representatives. M, molecular weight marker; S, the soluble fraction of the cell extract; I, the insoluble fraction of the cell extract; ST, Strep-Tactin affinity chromatography; Ni, Ni-NTA affinity chromatography; and TEV, cleavage with TEV protease. The protein sizes before and after TEV-protease treatment are indicated at the bottom.

Article Snippet: The resultant homogenous preparation was then treated with 6xHis-tagged TEV protease (MCLAB) at 4 °C for 24 h or longer if necessary, in order to cleave the 6xHis- and Twin-Strep-tags.

Techniques: Purification, Molecular Weight, Marker, Affinity Chromatography