e coli strain bl21 gold  (New England Biolabs)

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic representation of the pipeline for growth factor candidate selection and cell-based testing. (b) Western blot analysis of 11 high-value CFPS-derived growth factors. FGF-1, FGF-2, FGF-10, TNF-α, IL-1β, IFN-γ, and IL-15 were expressed in E. coli BL21 (DE3) in-house cell-free lysates. IL-6, EGF, IGF-1, and IL-3 were expressed in SHuffle-based in-house cell-free lysates. Detection was performed using an anti-His- HRP antibody. Data shown are from one representative biological replicate out of three independent experiments. (c) In vitro proliferation assay comparing on-demand, locally produced in Canada (blue) and commercial (green) FGF-1 growth factors in NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of growth factor. Luminescence was measured using a conventional plate reader. Data are shown as mean ± SD, n = 3. (d) In vitro proliferation assay comparing on-demand, locally produced (blue) and commercial (green) IL-3 growth factors in TF-1 cells. Cells were individually treated with varying concentrations (0.025, 0.05, 0.1, 0.5, 1, and 5 ng/mL). This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (e) Growth factor expression and cell-based validation in a low-resource setting. In vitro proliferation assay using FGF-1 and NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of both in-house-produced (blue) and commercial (green) growth factors. This representative data was obtained using reagents produced on-site in Brazil. Relative fold proliferation was plotted as the fold-change compared to untreated, serum-starved cells under the same experimental conditions. Data are shown as mean ± SD, n = 3. Statistical differences were determined by two-way ANOVA with Šídák’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations are: ns, not significantly different; kDa, kilodaltons.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Selection, Western Blot, Derivative Assay, In Vitro, Proliferation Assay, Produced, Expressing, Biomarker Discovery

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic of the SARS-CoV-2 genome; the coding sequence for the SARS-CoV-2 nucleocapsid protein was optimized for translation in CFPS and ultimately used for Nuvax formulation. (b) Western blot analysis of CFPS-produced SARS-CoV-2 nucleocapsid protein exhibiting a distinct band at the expected molecular weight (49 kDa). The SARS-CoV-2 nucleocapsid protein from SinoBiologicals, a commercially available recombinant protein, was used as a positive control (Ctrl +). N (CFPS) refers to the antigen expressed using in-house CFPS reactions. The molecular weight ladder (in kilodaltons) is shown on the left. Data shown are from one representative biological replicate out of three independent experiments. (c) Endotoxin levels in CFPS-produced SARS-CoV-2 nucleocapsid protein. Three different strategies were used for antigen expression: 1) cell-free lysates made from E. coli BL21(DE3); 2) cell-free lysates from E. coli BL21(DE3) followed by endotoxin removal; 3) cell-free lysates prepared from ClearColi TM BL21(DE3), an engineered E. coli strain used to express endotoxin-free recombinant proteins, making it ideal for expression of therapeutic products. The dashed line indicates the standard guidelines for subunit-based vaccine formulations (<20 EU/mL). Data are shown as mean ± SD, n = 3. (d) Immunization schedule, indicating prime, boost and bleed timepoints, for the CFPS-derived Nuvax. Intramuscular (IM) vaccination was administered on days 0 (prime) and 15 (boost), and blood was drawn on days 7, 14, 21, and 28 to evaluate for induction of IgG production. (e) Robust neutralizing antibody response following Nuvax administration alongside group controls. Analysis was performed using ELISA and measured at OD 450 nm using a conventional plate reader. The data are presented as the mean ± SD for each group, with individual data points shown (control group that received only PBS, n = 5; control group that received only adjuvants, n = 5; Nuvax group, n = 5). Statistical differences were determined by two-way ANOVA with Tukey’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations are: ns, not significantly different; Ctrl, control; N, nucleocapsid; End, endotoxin; Ab, antibody; IM, intramuscular; CFPS, cell-free protein synthesis.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Sequencing, Formulation, Western Blot, Produced, Molecular Weight, Recombinant, Positive Control, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic representation of the pipeline for growth factor candidate selection and cell-based testing. (b) Western blot analysis of 11 high-value CFPS-derived growth factors. FGF-1, FGF-2, FGF-10, TNF-α, IL-1β, IFN-γ, and IL-15 were expressed in E. coli BL21 (DE3) in-house cell-free lysates. IL-6, EGF, IGF-1, and IL-3 were expressed in SHuffle-based in-house cell-free lysates. Detection was performed using an anti-His- HRP antibody. Data shown are from one representative biological replicate out of three independent experiments. (c) In vitro proliferation assay comparing on-demand, locally produced in Canada (blue) and commercial (green) FGF-1 growth factors in NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of growth factor. Luminescence was measured using a conventional plate reader. Data are shown as mean ± SD, n = 3. (d) In vitro proliferation assay comparing on-demand, locally produced (blue) and commercial (green) IL-3 growth factors in TF-1 cells. Cells were individually treated with varying concentrations (0.025, 0.05, 0.1, 0.5, 1, and 5 ng/mL). This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (e) Growth factor expression and cell-based validation in a low-resource setting. In vitro proliferation assay using FGF-1 and NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of both in-house-produced (blue) and commercial (green) growth factors. This representative data was obtained using reagents produced on-site in Brazil. Relative fold proliferation was plotted as the fold-change compared to untreated, serum-starved cells under the same experimental conditions. Data are shown as mean ± SD, n = 3. Statistical differences were determined by two-way ANOVA with Šídák’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations are: ns, not significantly different; kDa, kilodaltons.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Selection, Western Blot, Derivative Assay, In Vitro, Proliferation Assay, Produced, Expressing, Biomarker Discovery

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic of the SARS-CoV-2 genome; the coding sequence for the SARS-CoV-2 nucleocapsid protein was optimized for translation in CFPS and ultimately used for Nuvax formulation. (b) Western blot analysis of CFPS-produced SARS-CoV-2 nucleocapsid protein exhibiting a distinct band at the expected molecular weight (49 kDa). The SARS-CoV-2 nucleocapsid protein from SinoBiologicals, a commercially available recombinant protein, was used as a positive control (Ctrl +). N (CFPS) refers to the antigen expressed using in-house CFPS reactions. The molecular weight ladder (in kilodaltons) is shown on the left. Data shown are from one representative biological replicate out of three independent experiments. (c) Endotoxin levels in CFPS-produced SARS-CoV-2 nucleocapsid protein. Three different strategies were used for antigen expression: 1) cell-free lysates made from E. coli BL21(DE3); 2) cell-free lysates from E. coli BL21(DE3) followed by endotoxin removal; 3) cell-free lysates prepared from ClearColi TM BL21(DE3), an engineered E. coli strain used to express endotoxin-free recombinant proteins, making it ideal for expression of therapeutic products. The dashed line indicates the standard guidelines for subunit-based vaccine formulations (<20 EU/mL). Data are shown as mean ± SD, n = 3. (d) Immunization schedule, indicating prime, boost and bleed timepoints, for the CFPS-derived Nuvax. Intramuscular (IM) vaccination was administered on days 0 (prime) and 15 (boost), and blood was drawn on days 7, 14, 21, and 28 to evaluate for induction of IgG production. (e) Robust neutralizing antibody response following Nuvax administration alongside group controls. Analysis was performed using ELISA and measured at OD 450 nm using a conventional plate reader. The data are presented as the mean ± SD for each group, with individual data points shown (control group that received only PBS, n = 5; control group that received only adjuvants, n = 5; Nuvax group, n = 5). Statistical differences were determined by two-way ANOVA with Tukey’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations are: ns, not significantly different; Ctrl, control; N, nucleocapsid; End, endotoxin; Ab, antibody; IM, intramuscular; CFPS, cell-free protein synthesis.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Sequencing, Formulation, Western Blot, Produced, Molecular Weight, Recombinant, Positive Control, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic representation of the pipeline for growth factor candidate selection and cell-based testing. (b) Western blot analysis of 11 high-value CFPS-derived growth factors. FGF-1, FGF-2, FGF-10, TNF-α, IL-1β, IFN-γ, and IL-15 were expressed in E. coli BL21 (DE3) in-house cell-free lysates. IL-6, EGF, IGF-1, and IL-3 were expressed in SHuffle-based in-house cell-free lysates. Detection was performed using an anti-His- HRP antibody. Data shown are from one representative biological replicate out of three independent experiments. (c) In vitro proliferation assay comparing on-demand, locally produced in Canada (blue) and commercial (green) FGF-1 growth factors in NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of growth factor. Luminescence was measured using a conventional plate reader. Data are shown as mean ± SD, n = 3. (d) In vitro proliferation assay comparing on-demand, locally produced (blue) and commercial (green) IL-3 growth factors in TF-1 cells. Cells were individually treated with varying concentrations (0.025, 0.05, 0.1, 0.5, 1, and 5 ng/mL). This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (e) Growth factor expression and cell-based validation in a low-resource setting. In vitro proliferation assay using FGF-1 and NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of both in-house-produced (blue) and commercial (green) growth factors. This representative data was obtained using reagents produced on-site in Brazil. Relative fold proliferation was plotted as the fold-change compared to untreated, serum-starved cells under the same experimental conditions. Data are shown as mean ± SD, n = 3. Statistical differences were determined by two-way ANOVA with Šídák’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations are: ns, not significantly different; kDa, kilodaltons.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Selection, Western Blot, Derivative Assay, In Vitro, Proliferation Assay, Produced, Expressing, Biomarker Discovery

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic of the SARS-CoV-2 genome; the coding sequence for the SARS-CoV-2 nucleocapsid protein was optimized for translation in CFPS and ultimately used for Nuvax formulation. (b) Western blot analysis of CFPS-produced SARS-CoV-2 nucleocapsid protein exhibiting a distinct band at the expected molecular weight (49 kDa). The SARS-CoV-2 nucleocapsid protein from SinoBiologicals, a commercially available recombinant protein, was used as a positive control (Ctrl +). N (CFPS) refers to the antigen expressed using in-house CFPS reactions. The molecular weight ladder (in kilodaltons) is shown on the left. Data shown are from one representative biological replicate out of three independent experiments. (c) Endotoxin levels in CFPS-produced SARS-CoV-2 nucleocapsid protein. Three different strategies were used for antigen expression: 1) cell-free lysates made from E. coli BL21(DE3); 2) cell-free lysates from E. coli BL21(DE3) followed by endotoxin removal; 3) cell-free lysates prepared from ClearColi TM BL21(DE3), an engineered E. coli strain used to express endotoxin-free recombinant proteins, making it ideal for expression of therapeutic products. The dashed line indicates the standard guidelines for subunit-based vaccine formulations (<20 EU/mL). Data are shown as mean ± SD, n = 3. (d) Immunization schedule, indicating prime, boost and bleed timepoints, for the CFPS-derived Nuvax. Intramuscular (IM) vaccination was administered on days 0 (prime) and 15 (boost), and blood was drawn on days 7, 14, 21, and 28 to evaluate for induction of IgG production. (e) Robust neutralizing antibody response following Nuvax administration alongside group controls. Analysis was performed using ELISA and measured at OD 450 nm using a conventional plate reader. The data are presented as the mean ± SD for each group, with individual data points shown (control group that received only PBS, n = 5; control group that received only adjuvants, n = 5; Nuvax group, n = 5). Statistical differences were determined by two-way ANOVA with Tukey’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations are: ns, not significantly different; Ctrl, control; N, nucleocapsid; End, endotoxin; Ab, antibody; IM, intramuscular; CFPS, cell-free protein synthesis.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Sequencing, Formulation, Western Blot, Produced, Molecular Weight, Recombinant, Positive Control, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular cell

Article Title: Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

doi: 10.1016/j.molcel.2024.06.002

Figure Lengend Snippet: Figure 1. Stalled ribosome:nascent chain complexes selectively recruit Trigger factor (A) Domain organization and structure of E. coli b-galactosidase (b-gal). Monomer (left) and homotetramer (right) colored by domain (PDB: 6CVM51). (B) b-gal ribosome:nascent chain complex (RNC) constructs. (C) RNC purification scheme. (D) Coomassie-stained SDS-PAGE of b-gal RNCs illustrated in (B) and purified as in (C) via two 1 M KOAc sucrose cushions. Bands corresponding to nascent chains (*) migrate higher (by 20 kDa) than expected based on protein molecular weight due to the covalently bound tRNA. (-) Released b-gal co-purifying with the RNCFL + 50G/S. Trigger factor (TF) and ribosomal protein S1 are indicated. The last lane contains purified full-length b-gal. Figure shows lanes from two in- dependent gels. Removal of unnecessary gel lanes is indicated by a dashed black line. (E) Intensity-based absolute quantification (iBAQ) of TF co-purified with RNCs in low- or high-salt. Values are normalized to the average iBAQ value of all 70S ribosomal proteins. TF was present at low (B) or high (C) levels in both purification conditions or exhibited salt-sensitive behavior (H). Where shown, error bars correspond to SD of 3 technical replicates. See also Figure S1 and Tables S1, S2, and S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-DnaK Enzo Life Sciences Cat#ADI-SPA-880-D, RRID:AB_2039064 Rabbit polyclonal anti-DnaJ Enzo Life Sciences Cat#ADI-SPA-410-D, RRID:AB_2039063 Mouse monoclonal anti-GroEL Abcam Cat#ab82592, RRID:AB_1658428 Rabbit polyclonal anti-S2 30S ribosomal protein Antibodies-Online Cat#ABIN2938988, RRID:AB_3076359 Rabbit polyclonal anti-TF GenScript Cat#A01329, RRID:AB_1575887 Mouse monoclonal anti-polyHistidine Merck Cat#A7058, RRID:AB_258326 HRP-conjugated goat polyclonal anti-rabbit Abcam Cat#ab205718, RRID:AB_2819160 HRP-conjugated goat polyclonal anti-mouse Abcam Cat#ab205719, RRID:AB_2755049 Bacterial and virus strains 5-alpha Competent E. coli NEB Cat#C2987 BL21 (DE3) E. coli NEB Cat#C2527 BL21-Gold-Dlac (DE3) E. coli Jeff Hasty, Didovyk et al.89 Addgene plasmid #99247 BL21 (DE3) DTF E. coli John Christodoulou, UCL N/A Chemicals, peptides, and recombinant proteins cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat#11873580001 Puromycin dihydrochloride Santa Cruz Biotechnology Cat#sc-108071 Benzonase Millipore Cat#E1014 RNase-free DNase QIAGEN Cat#79254 Proteinase K Millipore Cat#70663 o-nitrophenyl-b-D-galactopyranoside (o-NPG) ThermoScientific Cat#34055 PureLink Genomic DNA Mini Kit ThermoScientific Cat#K182000 Q5 Site-Directed Mutagenesis Kit NEB Cat#E0554 Gibson Assembly Master Mix NEB Cat#E2611 Phusion High-Fidelity DNA Polymerase NEB Cat#M0530 RiboLock RNase Inhibitor ThermoScientific Cat#EO0384 Halt Protease Inhibitor Cocktail (100X) ThermoScientific Cat#78437 Fluorescein-5-Maleimide ThermoScientific Cat#62245 DSBU ThermoScientific Cat#A35459 Hydroxylamine Merck Cat#467804 Monarch RNase A NEB Cat#T3018L BADAN Santa Cruz Biotechnology Cat#sc-210484 SuperSignal West Pico PLUS Chemiluminescent Substrate ThermoScientific Cat#34080 Sequencing Grade Modified Trypsin Promega Cat#V5111 Quick Coomassie Stain Neo Biotech Cat#NB-45-00078 Pepsin from porcine gastric mucosa Merck Cat#P6887 [Glu1]-Fibrinopeptide B Merck Cat#F3261 Deposited data HDX-MS analysis of RNCs This paper PRIDE: PXD048642 HDX-MS analysis of b-gal truncations This paper PRIDE: PXD048638 MS analysis of RNC composition This paper PRIDE: PXD048645 XL-MS This paper PRIDE: PXD048623 (Continued on next page) e1 Molecular Cell 84, 2455–2471.e1–e8, July 11, 2024

Techniques: Construct, Staining, SDS Page, Molecular Weight

Journal: Molecular cell

Article Title: Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

doi: 10.1016/j.molcel.2024.06.002

Figure Lengend Snippet: Figure 4. Structural determinants of TF bind- ing to nascent b-gal (A) TF binding to mutated RNCs. Coomassie- stained SDS-PAGE of the resuspended ribosomal pellet from high-salt co-sedimentation assays of RNCs, purified from WT E. coli, and incubated with additional TF in vitro. Bands corresponding to NCs (*) and TF are indicated. Immunoblots against TF are shown below. Positions of mutations are shown in red on the domain structure of NCs. (B–F) Conformational dynamics of b-gal truncations. Fractional deuteration difference, after 100 s deute- rium exposure, between full-length b-gal and b-gal chains truncated after 490 residues (B), 725 residues (C), 332 residues (D), 725 residues with an additional V567D mutation (E), or 440 residues (F). Darker red indicates more deuteration in the truncated chains compared with full-length b-gal. Orphaned domain interfaces are highlighted. Residue V567 is shown as cyan spheres. See also Figure S7 and Table S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-DnaK Enzo Life Sciences Cat#ADI-SPA-880-D, RRID:AB_2039064 Rabbit polyclonal anti-DnaJ Enzo Life Sciences Cat#ADI-SPA-410-D, RRID:AB_2039063 Mouse monoclonal anti-GroEL Abcam Cat#ab82592, RRID:AB_1658428 Rabbit polyclonal anti-S2 30S ribosomal protein Antibodies-Online Cat#ABIN2938988, RRID:AB_3076359 Rabbit polyclonal anti-TF GenScript Cat#A01329, RRID:AB_1575887 Mouse monoclonal anti-polyHistidine Merck Cat#A7058, RRID:AB_258326 HRP-conjugated goat polyclonal anti-rabbit Abcam Cat#ab205718, RRID:AB_2819160 HRP-conjugated goat polyclonal anti-mouse Abcam Cat#ab205719, RRID:AB_2755049 Bacterial and virus strains 5-alpha Competent E. coli NEB Cat#C2987 BL21 (DE3) E. coli NEB Cat#C2527 BL21-Gold-Dlac (DE3) E. coli Jeff Hasty, Didovyk et al.89 Addgene plasmid #99247 BL21 (DE3) DTF E. coli John Christodoulou, UCL N/A Chemicals, peptides, and recombinant proteins cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat#11873580001 Puromycin dihydrochloride Santa Cruz Biotechnology Cat#sc-108071 Benzonase Millipore Cat#E1014 RNase-free DNase QIAGEN Cat#79254 Proteinase K Millipore Cat#70663 o-nitrophenyl-b-D-galactopyranoside (o-NPG) ThermoScientific Cat#34055 PureLink Genomic DNA Mini Kit ThermoScientific Cat#K182000 Q5 Site-Directed Mutagenesis Kit NEB Cat#E0554 Gibson Assembly Master Mix NEB Cat#E2611 Phusion High-Fidelity DNA Polymerase NEB Cat#M0530 RiboLock RNase Inhibitor ThermoScientific Cat#EO0384 Halt Protease Inhibitor Cocktail (100X) ThermoScientific Cat#78437 Fluorescein-5-Maleimide ThermoScientific Cat#62245 DSBU ThermoScientific Cat#A35459 Hydroxylamine Merck Cat#467804 Monarch RNase A NEB Cat#T3018L BADAN Santa Cruz Biotechnology Cat#sc-210484 SuperSignal West Pico PLUS Chemiluminescent Substrate ThermoScientific Cat#34080 Sequencing Grade Modified Trypsin Promega Cat#V5111 Quick Coomassie Stain Neo Biotech Cat#NB-45-00078 Pepsin from porcine gastric mucosa Merck Cat#P6887 [Glu1]-Fibrinopeptide B Merck Cat#F3261 Deposited data HDX-MS analysis of RNCs This paper PRIDE: PXD048642 HDX-MS analysis of b-gal truncations This paper PRIDE: PXD048638 MS analysis of RNC composition This paper PRIDE: PXD048645 XL-MS This paper PRIDE: PXD048623 (Continued on next page) e1 Molecular Cell 84, 2455–2471.e1–e8, July 11, 2024

Techniques: Binding Assay, Staining, SDS Page, Sedimentation, Incubation, In Vitro, Western Blot, Mutagenesis, Residue

Journal: Molecular cell

Article Title: Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

doi: 10.1016/j.molcel.2024.06.002

Figure Lengend Snippet: Figure 5. Architecture of complexes between DnaK and RNCs (A) Binding of DnaJ/K to RNCs. Left: co-sedimentation assay. Empty ribosomes or RNCs purified from DTF E. coli were incubated with DnaK, co-chaperones, and ATP, then centrifuged through a sucrose cushion to separate the ribosomal fraction (pellet) from unbound (co-)chaperones (supernatant). Right: Coomassie- stained SDS-PAGE of the resuspended ribosomal pellet. RNC1–646 was incubated with either DnaK (+K), DnaJ (+J), both DnaK and DnaJ (+KJ), or DnaK, DnaJ, and GrpE (+KJ+E), with or without ATP as indicated. An immunoblot against DnaJ is shown below. (B) Client processing by the Hsp70 system. DnaJ and GrpE control the transition of DnaK between conformations with low (ATP state) and high (ADP state) affinity for the client protein. (C) XL-MS of DnaK:RNC complexes. Top: number of unique crosslinks between each DnaK domain (NBD: purple, SBD: pink) and the NC of different b-gal RNCs. Bottom: crosslinks between DnaK and RNC1–646. (D) Number of unique crosslinks between DnaK and residues on each NC, grouped according to their position in b-gal domains as in Figure 3A. (E) Cysteine-painting analysis of b-gal conformation. Error bars correspond to the SD of 3 independent labeling reactions. (F) Effect of DnaK on NC conformation. Top: difference in deuterium uptake, after 10 or 100 s deuteration, between NC peptides in RNC1–646 with or without bound DnaK. Lower values indicate less deuteration of peptides in DnaK-bound RNC1–646. Bottom: regions showing changes in deuterium uptake R0.5 Da at either time point are mapped onto the structure of b-gal truncated after 626 residues. Residues on RNC1–646 that crosslink to DnaK are shown as orange spheres. Regions that were not covered in the experiment are colored dark gray. See also Figures S3, S8, and S9 and Tables S5 and S7–S9.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-DnaK Enzo Life Sciences Cat#ADI-SPA-880-D, RRID:AB_2039064 Rabbit polyclonal anti-DnaJ Enzo Life Sciences Cat#ADI-SPA-410-D, RRID:AB_2039063 Mouse monoclonal anti-GroEL Abcam Cat#ab82592, RRID:AB_1658428 Rabbit polyclonal anti-S2 30S ribosomal protein Antibodies-Online Cat#ABIN2938988, RRID:AB_3076359 Rabbit polyclonal anti-TF GenScript Cat#A01329, RRID:AB_1575887 Mouse monoclonal anti-polyHistidine Merck Cat#A7058, RRID:AB_258326 HRP-conjugated goat polyclonal anti-rabbit Abcam Cat#ab205718, RRID:AB_2819160 HRP-conjugated goat polyclonal anti-mouse Abcam Cat#ab205719, RRID:AB_2755049 Bacterial and virus strains 5-alpha Competent E. coli NEB Cat#C2987 BL21 (DE3) E. coli NEB Cat#C2527 BL21-Gold-Dlac (DE3) E. coli Jeff Hasty, Didovyk et al.89 Addgene plasmid #99247 BL21 (DE3) DTF E. coli John Christodoulou, UCL N/A Chemicals, peptides, and recombinant proteins cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat#11873580001 Puromycin dihydrochloride Santa Cruz Biotechnology Cat#sc-108071 Benzonase Millipore Cat#E1014 RNase-free DNase QIAGEN Cat#79254 Proteinase K Millipore Cat#70663 o-nitrophenyl-b-D-galactopyranoside (o-NPG) ThermoScientific Cat#34055 PureLink Genomic DNA Mini Kit ThermoScientific Cat#K182000 Q5 Site-Directed Mutagenesis Kit NEB Cat#E0554 Gibson Assembly Master Mix NEB Cat#E2611 Phusion High-Fidelity DNA Polymerase NEB Cat#M0530 RiboLock RNase Inhibitor ThermoScientific Cat#EO0384 Halt Protease Inhibitor Cocktail (100X) ThermoScientific Cat#78437 Fluorescein-5-Maleimide ThermoScientific Cat#62245 DSBU ThermoScientific Cat#A35459 Hydroxylamine Merck Cat#467804 Monarch RNase A NEB Cat#T3018L BADAN Santa Cruz Biotechnology Cat#sc-210484 SuperSignal West Pico PLUS Chemiluminescent Substrate ThermoScientific Cat#34080 Sequencing Grade Modified Trypsin Promega Cat#V5111 Quick Coomassie Stain Neo Biotech Cat#NB-45-00078 Pepsin from porcine gastric mucosa Merck Cat#P6887 [Glu1]-Fibrinopeptide B Merck Cat#F3261 Deposited data HDX-MS analysis of RNCs This paper PRIDE: PXD048642 HDX-MS analysis of b-gal truncations This paper PRIDE: PXD048638 MS analysis of RNC composition This paper PRIDE: PXD048645 XL-MS This paper PRIDE: PXD048623 (Continued on next page) e1 Molecular Cell 84, 2455–2471.e1–e8, July 11, 2024

Techniques: Binding Assay, Sedimentation, Incubation, Staining, SDS Page, Western Blot, Control, Structural Proteomics, Labeling

Journal: Molecular cell

Article Title: Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

doi: 10.1016/j.molcel.2024.06.002

Figure Lengend Snippet: Figure 6. DnaJ binds RNCs using an extensive surface spread across multiple domains (A) Domain organization and predicted structure (AF-P08622-F1) of E. coli DnaJ monomer. J domain (JD, dark blue), G/F-rich region (G/F, gray), zinc-binding domain (ZBD, white), C-terminal b-sandwich domains (CTD I, aquamarine, and CTD II, cyan), and dimerization domain (DD, light blue). (B) XL-MS of DnaJ:RNC complexes. Number of unique crosslinks between each DnaJ domain and the NC of different b-gal RNCs. (C) Crosslink sites on DnaJ. AlphaFold2.0 multimer-predicted structure of dimeric DnaJ, colored as in (A) with residues that crosslink to any tested NC shown as orange spheres. (D) NC-binding sites on DnaJ. Difference in deuterium uptake between isolated DnaJ and DnaJ bound to RNC1–646. Regions showing changes in deuterium uptake R0.5 Da (after either 10 or 100 s deuteration) are mapped onto the structure of DnaJ. Darker blue indicates less deuteration of RNC-bound DnaJ. Residues on DnaJ that crosslink to NC1–646 are colored orange. Regions that were not covered in the experiment are colored dark gray. (E) Effect of DnaJ on NC1–646 conformation. Top: difference in deuterium uptake, after 10 or 100 s deuteration, between NC peptides in RNC1–646 with or without bound DnaJ. Lower values indicate less deuteration of peptides in DnaJ-bound RNC1–646 relative to free RNC1–646. Bottom: b-gal monomer structure truncated after 626 residues, with protected regions (R0.5 Da) colored blue, sites detected in DnaJ peptide array colored green, and residues that crosslink to DnaJ shown as orange spheres. Regions that were not covered in the HDX-MS experiment are colored dark gray. (F) Model of DnaJ binding to RNCs and recruitment of DnaK. See also Figures S3 and S10 and Tables S5 and S8.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-DnaK Enzo Life Sciences Cat#ADI-SPA-880-D, RRID:AB_2039064 Rabbit polyclonal anti-DnaJ Enzo Life Sciences Cat#ADI-SPA-410-D, RRID:AB_2039063 Mouse monoclonal anti-GroEL Abcam Cat#ab82592, RRID:AB_1658428 Rabbit polyclonal anti-S2 30S ribosomal protein Antibodies-Online Cat#ABIN2938988, RRID:AB_3076359 Rabbit polyclonal anti-TF GenScript Cat#A01329, RRID:AB_1575887 Mouse monoclonal anti-polyHistidine Merck Cat#A7058, RRID:AB_258326 HRP-conjugated goat polyclonal anti-rabbit Abcam Cat#ab205718, RRID:AB_2819160 HRP-conjugated goat polyclonal anti-mouse Abcam Cat#ab205719, RRID:AB_2755049 Bacterial and virus strains 5-alpha Competent E. coli NEB Cat#C2987 BL21 (DE3) E. coli NEB Cat#C2527 BL21-Gold-Dlac (DE3) E. coli Jeff Hasty, Didovyk et al.89 Addgene plasmid #99247 BL21 (DE3) DTF E. coli John Christodoulou, UCL N/A Chemicals, peptides, and recombinant proteins cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat#11873580001 Puromycin dihydrochloride Santa Cruz Biotechnology Cat#sc-108071 Benzonase Millipore Cat#E1014 RNase-free DNase QIAGEN Cat#79254 Proteinase K Millipore Cat#70663 o-nitrophenyl-b-D-galactopyranoside (o-NPG) ThermoScientific Cat#34055 PureLink Genomic DNA Mini Kit ThermoScientific Cat#K182000 Q5 Site-Directed Mutagenesis Kit NEB Cat#E0554 Gibson Assembly Master Mix NEB Cat#E2611 Phusion High-Fidelity DNA Polymerase NEB Cat#M0530 RiboLock RNase Inhibitor ThermoScientific Cat#EO0384 Halt Protease Inhibitor Cocktail (100X) ThermoScientific Cat#78437 Fluorescein-5-Maleimide ThermoScientific Cat#62245 DSBU ThermoScientific Cat#A35459 Hydroxylamine Merck Cat#467804 Monarch RNase A NEB Cat#T3018L BADAN Santa Cruz Biotechnology Cat#sc-210484 SuperSignal West Pico PLUS Chemiluminescent Substrate ThermoScientific Cat#34080 Sequencing Grade Modified Trypsin Promega Cat#V5111 Quick Coomassie Stain Neo Biotech Cat#NB-45-00078 Pepsin from porcine gastric mucosa Merck Cat#P6887 [Glu1]-Fibrinopeptide B Merck Cat#F3261 Deposited data HDX-MS analysis of RNCs This paper PRIDE: PXD048642 HDX-MS analysis of b-gal truncations This paper PRIDE: PXD048638 MS analysis of RNC composition This paper PRIDE: PXD048645 XL-MS This paper PRIDE: PXD048623 (Continued on next page) e1 Molecular Cell 84, 2455–2471.e1–e8, July 11, 2024

Techniques: Binding Assay, Structural Proteomics, Isolation, Peptide Microarray