Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: β-gal structure. Top, organisation of domains 1-5 (D1-5). Left, monomer structure (PDB: 6CVM ). Right, tetramer structure showing long and activating interfaces. b, Refolding of β-gal upon dilution from 8 M urea into buffer at 10 or 37 °C, assayed by recovery of enzyme activity. Data were fit to a single exponential function. Yield = 25.1%; 95% CI 24.5-25.7%. Half-time = 4.3 h; 95% CI 3.9-4.6 h. Error bars represent s.d. (n = 3). c, Specific activity of β-gal purified from E. coli (n=8 technical replicates) or produced by in vitro translation (IVT) (n=3 independent IVT reactions). Data were normalised to the mean specific activity of purified β-gal. Error bars represent s.d. d, Amount and activity of in vitro translated full-length β-gal, at different times after initiating IVTs using a plasmid encoding β-gal. Error bars represent s.d. (n = 3 independent IVT reactions). e, Accumulation of β-gal activity during IVT reaction. After 40 min (arrow), reactions were treated with 5 mM chloramphenicol (CAM), 5 mM puromycin (PURO), or an equal volume of water (H 2 O). f, β-gal activity in ribosomal fraction of IVTs. β-gal was expressed for 1 h before supplementing the reaction with either CAM to stabilise ribosome-nascent chain complexes, or PURO to release nascent chains. The ribosomal fraction was isolated by sucrose cushion ultracentrifugation. Error bars represent s.d. (n = 3 independent IVT reactions). ns - p>0.05; * - p<0.05 based on one-way ANOVA with Dunnett’s multiple comparisons. g, Oligomeric state of natively folded (native), refolded, or IVT-produced β-gal was analysed using native-PAGE. Duplicate gels were immunoblotted for β-gal or stained with the β-gal substrate X-gal. Bands corresponding to the tetramer (T), dimer (D), monomer (M) or intermediate (I) are indicated.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Activity Assay, Purification, Produced, In Vitro, Plasmid Preparation, Isolation, Clear Native PAGE, Staining

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: β-gal refolding upon dilution from 8 M urea into buffer at different temperatures, assayed by recovery of enzyme activity. Data were fit to a single exponential function. Error bars represent s.d. (n = 3). b, Arrhenius plot showing temperature dependence of β-gal refolding kinetics as measured in ( a ). E a = 84±1 kJ.mol -1 . Error bars represent s.d. (n = 3). c, Influence of Hsp70 chaperone system on β-gal refolding. Refolding was measured as in (a), at 37 °C. The reaction was supplemented with purified DnaK, DnaJ and GrpE, with or without ATP. Data were fit to a single exponential function, giving t ½ = 11 min (95% CI, 8 – 14 min) in the presence of ATP. Error bars represent s.d. (n = 3). d, Aggregation during β-gal refolding. Turbidity was monitored by absorbance at 320 nm. Refolding under optimal conditions as in (10 °C, final urea concentration of 1.5 M) did not result in detectable turbidity. Turbidity could be induced by raising the temperature of the reaction to 25 °C and reducing the final urea concentration to 0.16 M). The shaded region represents s.d. (n = 3). e , Oligomeric state of β-gal during refolding, analysed using native-PAGE. Replicate gels were stained with Coomassie, immunoblotted for β-gal, or stained with the β-gal substrate X-gal. Bands corresponding to the tetramer (T), dimer (D), monomer (M) or intermediate (I) are indicated. Natively folded β-gal purified from E.coli is included as a control.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Activity Assay, Purification, Concentration Assay, Clear Native PAGE, Staining, Control

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: Coomassie-stained gel of RNCs purified from E.coli lacking endogenous Trigger factor (Δ tig ) or β-gal (Δ lacZ ). Ribosomal proteins (r.p.), Trigger factor (TF), the nascent chain linked to its peptidyl-tRNA (*) and co-purifying full-length β-gal (**) are indicated. Purified β-gal and empty 70S ribosomes are shown alongside for reference.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Staining, Purification

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: β-gal enzyme activity of 5 nM RNCs purified from wild-type E. coli . Error bars represent s.d. (n = 3). b, Endogenous β-gal assembles with RNCs. Enzyme activity of 5 nM RNCs purified from wild-type, or Δ lacZ E. coli which do not express β-gal. Error bars represent s.d. (n = 3). c, Onset of co-post β-gal assembly in vivo. SeRP data showing the codon-resolved enrichment (selected translatome / total translatome) of β-gal-bound RNCs along the lacZ open reading frame. d, D1-4 are active off but not on the ribosome. Enzyme activity of 120 nM 1-725 β-gal truncation (domains 1-4) and RNC 1-745 , both purified from Δ lacZ E. coli . Error bars represent s.d. (n = 3). e, Truncated β-gal misassembles. Negative-stain EM micrograph of β-gal 1-725 purified from Δ lacZ E. coli. Particles corresponding to tetramers or higher-order oligomers are indicated with blue or white circles, respectively. f, β-gal assembly is triggered by folded D5. Enzyme activity of 7.5 nM β-gal RNCs purified from Δ lacZ E. coli , in the absence or presence of isolated folded domain 5 (D5, residues 739-1023). The distance between D1-4 (residues 1-725) and the ribosome was increased by inserting a 50 or 100 aa Gly-Ser linker between the C-terminus of the NC and the stalling sequence. g, D5 folds primarily post-translation. Difference in deuterium uptake, after 100 s deuteration, between native β-gal and RNC 1-1014 purified from Δ lacZ E. coli . Regions that are deprotected in the RNC are coloured in shades of red. The C-terminal 30 aa, expected to be inside the exit tunnel in RNC 1-1014 , are coloured green.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Activity Assay, Purification, In Vivo, Staining, Isolation, Sequencing

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: Isolated RNCs are inactive. Stalling constructs were expressed in PURE IVT reactions as in . Enzyme activity was monitored in real-time by fluorescence upon cleavage of the substrate resorufin-β-D-glucopyranoside. Full-length β-gal (with a stop codon) was translated as a control. b , Copurification of endogenous full-length β-gal with RNC 1-1014 . Wild-type RNC 1-1014 (RNC 1-1014WT ) or a variant with a disrupted activation interface (RNC 1-1014Δ11-41 ) were purified from wild-type (WT), Δ lacZ or Δ tig E. coli and resolved by SDS-PAGE followed by Coomassie staining. The last lane contains purified full-length β-gal for reference. Bands corresponding to the NCs (*), copurifying full-length (**) or C-terminally truncated (***) β-gal, Trigger factor (TF) and ribosomal proteins (r.p.) are indicated. Right: immunoblot of RNC 1-1014WT purified from wild-type cells, probed using an antibody against the β-gal N-terminus (ab106567). c , Copurifying full-length β-gal is protease-resistant compared to the NC. RNC 1-1014 purified from WT or Δ lacZ cells was treated with Proteinase K and resolved by SDS-PAGE followed by Coomassie staining. Bands corresponding to the NC (*) and to the full-length β-galactosidase (**) are indicated. d, β-gal pulldown specifically enriches lacZ mRNA. Comparison of the gene-specific footprint abundance in the β-galactosidase co-IP sample and the total translatome in RPKM (reads per kilobase per million mapped reads). The lacZ transcript is indicated in blue. e , 1-519 β-gal truncation is monomeric. Molecular weight was measured by SEC-MALS at different concentrations as indicated. Molecular weight (MW, left y-axis) and ultraviolet absorbance (UV, right y-axis) are plotted as a function of elution time. The theoretical mass of the 1-519 fragment is 59 kDa. f , lacZ -translating ribosomes are not enriched in the disome fraction. Comparison of the gene-specific footprint abundance in disome and monosome samples in RPKM (reads per kilobase per million mapped reads). The lacZ transcript is indicated in yellow. g, β-gal does not assemble via a co-co mechanism. Monosome (blue) and disome (yellow) footprint density along the lacZ open reading frame. RPM: reads per million. h , 1-725 β-gal truncation is partially tetrameric. SEC-MALS was performed as in ( d ), at a concentration of 30 μM. Two species of different molecular weights were detected, corresponding to monomeric (87 kDa) and tetrameric (320 kDa) forms. The theoretical mass of the 1-725 fragment is 83 kDa. i , Full-length β-gal forms tetramers rather than higher order assemblies as detected in for the 1-725 fragment. Negative stain electron microscopy micrograph of purified full-length β-gal. The scale bar corresponds to 50 nm.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Isolation, Construct, Activity Assay, Fluorescence, Control, Copurification, Variant Assay, Activation Assay, Purification, SDS Page, Staining, Western Blot, Comparison, Co-Immunoprecipitation Assay, Molecular Weight, Concentration Assay, Electron Microscopy

Journal: ACS Catalysis

Article Title: Ultrahigh-Throughput Activity Engineering of Promiscuous Amidases through a Fluorescence-Activated Cell Sorting Assay

doi: 10.1021/acscatal.5c01903

Figure Lengend Snippet: FACS-based detection of amidase activity. ( A ) Overview of the FACS-based assay principle. Fluorescence histograms of E. coli BL21­(DE3)-Gold cells expressing GST only as a negative control ( B ) or co-expressing GST and the amidase Pd Amd ( C ) or Sa Amd ( D ), respectively. Amidase activity in whole cells towards 1 was measured by flow cytometry (FL3-UV: λ ex = 375 nm/λ em = 440 nm); 1 and 1-GSH do not exhibit fluorescence.

Article Snippet: The following strains were ordered from Thermo Fisher Scientific: E. coli BL21 (DE3)-Gold and E. coli TOP10.

Techniques: Activity Assay, Fluorescence, Expressing, Negative Control, Flow Cytometry

Journal: Frontiers in Microbiology

Article Title: Detoxification of aflatoxin B1 by a Bacillus subtilis spore coat protein through formation of the main metabolites AFQ1 and epi-AFQ1

doi: 10.3389/fmicb.2024.1406707

Figure Lengend Snippet: Assessment of the genotoxic potential of AFB1 and AFQ1 by the SOS Chromotest based on the measurement of the primary response of E.coli cells to genetic damage. Estimated SOSIF values for AFB1 against AfQ1 resulting after decomposition by laccase. SOSIF, SOS induction factor.

Article Snippet: Chemically competent E. coli BL21-Gold (DE3) cells (Invitrogen, Carlsbad, CA, USA) were transformed with the plasmid and the laccase expression induced by addition of IPTG.

Techniques: SOS Chromotest