Journal: bioRxiv

Article Title: Optimizing protein expression in the One-Pot PURE System: Insights into reaction composition and translation efficiency

doi: 10.1101/2024.06.19.599772

Figure Lengend Snippet: (A) Schematic of protein expression and regulation in E. coli strains M15/pREP4 and BL21(DE3) for Dual Strain One-Pot PURE systems. Each gene of interest is expressed under the control of either the PT5 or PT7 promoter followed by lacO operator site(s). In the M15/pREP4 expression strain, the pREP4 plasmid constitutively expresses the lac inhibitor, LacI, which represses protein expression by binding to the lacO sites. In BL21(DE3), endogenous LacI represses the production of T7 RNA polymerase (RNAP) and gene expression by binding to the lacO sites. Addition of the IPTG inducer de-represses LacI inhibition and activates gene expression. (B) Schematic of monoculture protein expression workflow. Cells were grown in 96-well deep-well plates, and protein expression was activated by adding 0.1 mM IPTG inducer. Following cell growth, protein expression was assessed via SDS-PAGE. (C) Monoculture protein expression assessment from cells grown in LB media. Ten strains (designated by *) were found to carry the protein expression plasmid but lacked the protein expression capability. (D) Stable protein expression from cells grown in LB with 1% w/v glucose after 5 passages, with all PURE proteins visibly expressed.

Article Snippet: E. coli BL21(DE3) (Thermo Scientific) and M15/pREP4 (Qiagen) strains were used for protein expression.

Techniques: Expressing, Control, Plasmid Preparation, Binding Assay, Inhibition, SDS Page

Journal: bioRxiv

Article Title: Optimizing protein expression in the One-Pot PURE System: Insights into reaction composition and translation efficiency

doi: 10.1101/2024.06.19.599772

Figure Lengend Snippet: (A) Schematic of One-Pot PURE reaction composition using in-house prepared energy solutions with commercial tRNAs from E. coli MRE600 and E. coli Strain W. Each reaction contains 5 nM of PT7-UTR1-deGFP to assess protein production. (B) Comparison of deGFP protein production and its rate of production for two batches of Single Strain One-Pot PURE proteins (added to the reaction either at 2.5 or 5.0 mg/mL) in energy solution with 22 A260U/mL E. coli MRE600 tRNA. Error bars represent standard deviations of reaction triplicates. Protein translation rate plots represent the average of reaction triplicates. (C) Comparison of deGFP protein production and its rate of production for two batches of Single Strain One-Pot PURE proteins (added to the reaction at 2.5 mg/mL) in energy solution with increasing E. coli Strain W tRNA activity units (30 – 80 A260U/mL). The plots represent the average of reaction triplicates, and the error bars represent standard deviations of reaction triplicates. Protein translation rate plots represent the average of reaction triplicates.

Article Snippet: E. coli BL21(DE3) (Thermo Scientific) and M15/pREP4 (Qiagen) strains were used for protein expression.

Techniques: Comparison, Activity Assay

Journal: Molecular plant

Article Title: DET1-mediated COP1 regulation avoids HY5 activity over second-site gene targets to tune plant photomorphogenesis.

doi: 10.1016/j.molp.2021.03.009

Figure Lengend Snippet: Figure 1. DET1- and COP1-associated proteins. (A) Schematic representation of proteins found to associate with DET1 and COP1 in TAP assays. Color code represents the maximum number of peptides for each represented protein found in a TAP assay as detailed in Supplemental Table 1. DET1 and COP1 proteins were expressed in Arabidopsis cell cultures. Five independent TAP experiments were performed for DET1 and two for COP1 (Supplemental Table 2). (B) MBP-COP1 and MBP-HY5 recombinant proteins expressed in E. coli pulled-down MYC-DET1 from 7-day-old Arabidopsis seedlings. MBP re- combinant protein was used as a control. Anti-MYC and anti-MBP antibodies were used for the immunoblots. (C and D) F€orster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET–FLIM) analysis of the interaction between COP1 or HY5 fused to GFP and DET1 (C), and HY5 or COP1 fused to RFP (D). Box plots show the distribution of 5–9 measurements ±SD. (E) FRET–FLIM analysis of the interaction between GFP-COP1 and RFP-HY5 upon cluc-DET1 co-expression. Box plots show the distribution of 10 measurements ±SD. All FRET assays were performed following transient expression in N. benthamiana leaves. Free RFP was used as negative control. FE, FRET efficiency. Asterisks indicate statistically significant differences according to Student’s t-test (****p < 0.0001; ***p < 0.001; *p < 0.01). For all FRET experiments three independent biological replicates were performed, and results from one replicate are shown.

Article Snippet: Pull-down assays MBP recombinant protein fusions were expressed in the Escherichia coli BL21 (DE3) strain carrying the corresponding coding sequence cloned into the pKM596 plasmid, a gift from David Waugh (Addgene plasmid #8837).

Techniques: Recombinant, Control, Western Blot, Förster Resonance Energy Transfer, Imaging, Microscopy, Expressing, Negative Control