Journal: bioRxiv

Article Title: Computation-guided redesign of promoter specificity of a bacterial RNA polymerase

doi: 10.1101/2022.11.29.518332

Figure Lengend Snippet: ( a ), Interface scores of all (gray) or 1000 top scoring (red) σ70 variants in complex with each −35 DNA target. All single, double, triple, and quadruple combinatorial variants of σ70 positions 584, 585, 586, 588, and 589 were modeled using Rosetta. ( b ), Position-specific amino acid enrichment scores (red gradient) among selected top scoring σ70 variants. WT identity (boxed outline) and most enriched amino acid (*) at each mutable position. ( c ), Cartoon schematic showing H-bonds formed between each enriched σ70 consensus sequence and −35 DNA target in the Rosetta structural models.

Article Snippet: For Rosetta designed σ variants, 110-base pair (bp) single strand DNA oligo pool containing Rosetta designed σ fragments were ordered from Agilent.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Computation-guided redesign of promoter specificity of a bacterial RNA polymerase

doi: 10.1101/2022.11.29.518332

Figure Lengend Snippet: ( a ), Normalized fluorescence of WT σ70 (gray) and the top three performing variants (red, yellow, or blue) for each promoter target. Clones were selected from the sorted libraries ( Supplementary Fig. X ) and assayed in a 96-well fluorescence plate reader. Error bars denote the standard deviation (**P ≤ 0.01, ***P ≤ 0.001) of replicates (n≥3) and computed fold-changes are relative to WT. RFU, relative fluorescence units. ( b ), Cartoon schematic showing H-bonds formed between each σ70 variant and the cognate −35 DNA target in the Rosetta structural models.

Article Snippet: For Rosetta designed σ variants, 110-base pair (bp) single strand DNA oligo pool containing Rosetta designed σ fragments were ordered from Agilent.

Techniques: Fluorescence, Clone Assay, Standard Deviation, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Stabilized designs of the malaria adhesin protein PvRBP2b for use as a potential diagnostic for Plasmodium vivax

doi: 10.1016/j.jbc.2025.108290

Figure Lengend Snippet: Design process of stabilized PvRBP2b 169-470 variants. Stabilized designs of PvRBP2b were computed under stringent conditions of minimally perturbing the solvent-accessible surfaces to maintain its antigenicity profile. Due to limited diversity of natural PvRBP2b homologs, in some designs, we used AI-based ProteinMPNN to generate a pseudo-PSSM followed by PROSS atomistic design calculations. A total of nine designs were tested. PvRBP2b, Plasmodium vivax reticulocyte-binding protein 2b.

Article Snippet: Restriction enzyme cloning was used to clone the synthetic DNA fragments (obtained from Twist Biosciences) of PvRBP2b 169-470 designs into pPROEX HTb vector.

Techniques: Solvent, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Stabilized designs of the malaria adhesin protein PvRBP2b for use as a potential diagnostic for Plasmodium vivax

doi: 10.1016/j.jbc.2025.108290

Figure Lengend Snippet: Purification yields and biophysical characterization of parental PvRBP2b 169-470 and designs. A , Coomassie-stained SDS-PAGE gel of Ni-NTA affinity purified parental PvRBP2b 169-470 and nine designs in reducing conditions. B , final yields (mg/L bacterial culture) for parental and stabilized designs after Ni-NTA affinity, ion exchange, and size-exclusion purification steps with fold change increase relative to parental yields shown on top of the corresponding bar graphs. The dotted line separates the two independent replicates for protein purification. C , dynamic light scattering (DLS) measurements of parental and three stabilized designs showing the hydrodynamic radius ( R h ) and unfolding onset temperatures (T onset ) from two independent replicates. PvRBP2b, Plasmodium vivax reticulocyte-binding protein 2b.

Article Snippet: Restriction enzyme cloning was used to clone the synthetic DNA fragments (obtained from Twist Biosciences) of PvRBP2b 169-470 designs into pPROEX HTb vector.

Techniques: Purification, Staining, SDS Page, Affinity Purification, Protein Purification, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Stabilized designs of the malaria adhesin protein PvRBP2b for use as a potential diagnostic for Plasmodium vivax

doi: 10.1016/j.jbc.2025.108290

Figure Lengend Snippet: Structural comparison of parental PvRBP2b 169-470 with stabilized designs WHT2483 and 2484. A , crystal structures of WHT2483 ( purple ) and WHT2484 ( green ) are overlayed with parental PvRBP2b 169-470 with RMSD values provided. B , mutated residues are shown as spheres mapped onto ribbon representations of the parental PvRBP2b 169-470 structure. Mutations present in both WHT2483 and WHT2484 are shown in pink , and mutations present only in WHT2484 are shown in green . Representative mutations that demonstrate stabilising effects are highlighted on the tertiary structure compared with parental PvRBP2b 169-470 . C , binding footprints of human antibodies against parental PvRBP2b 169-470 . Parental PvRBP2b 169-470 is shown in surface representation ( white ), and coloured regions denote residues involved in antibody binding, with light chain interactions in a lighter shade and heavy chain interactions in a darker shade for each antibody. Interacting residues are obtained from previously published work and were determined using PISA . Mutations within an antibody binding footprint are shown in blue for those in WHT2482 to 2484 inclusive (241242 Q378L) and teal for WHT2484 alone (237235 K248L and 283284 M324F). D , total antibody bound surface ( deep blue ) for the eight human mAbs is shown on parental PvRBP2b 169-470 surface ( white ). PvRBP2b, Plasmodium vivax reticulocyte-binding protein 2b.

Article Snippet: Restriction enzyme cloning was used to clone the synthetic DNA fragments (obtained from Twist Biosciences) of PvRBP2b 169-470 designs into pPROEX HTb vector.

Techniques: Comparison, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Stabilized designs of the malaria adhesin protein PvRBP2b for use as a potential diagnostic for Plasmodium vivax

doi: 10.1016/j.jbc.2025.108290

Figure Lengend Snippet: Human antibody affinities to parental PvRBP2b 169-470 and three stabilized designs

Article Snippet: Restriction enzyme cloning was used to clone the synthetic DNA fragments (obtained from Twist Biosciences) of PvRBP2b 169-470 designs into pPROEX HTb vector.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Stabilized designs of the malaria adhesin protein PvRBP2b for use as a potential diagnostic for Plasmodium vivax

doi: 10.1016/j.jbc.2025.108290

Figure Lengend Snippet: Stabilized PvRBP2b 169-470 designs function as reliable serological markers for recent P. vivax infection. A , antibody responses toward PvRBP2b proteins measured in relative antibody units (RAUs) from the year-long cohort studies, as well as negative controls from Australian Red Cross (ARC), Brazil (Br Neg), Thai Red Cross (ThRC), and the Volunteer Blood Donor Registry (VBDR) in Victoria, Australia. Boxplots illustrate the median and 25th and 75th percentiles of the distribution of antibody responses for individuals who had a (i) current infection ( i.e. , positive qPCR results for P. vivax at the time of antibody measurement), (ii) recent infection within the previous 9 months, (iii) old infection ( i.e. , infection nine to 12 months ago), (iv) no infection during the year-long cohort study, and (v) the negative controls. B , correlation between parental PvRBP2b 169-470 and designs and associated Pearson correlation coefficient (R), with colors representing the infection status as indicated. C , receiver operating characteristic (ROC) curve for eight-antigen sero-diagnostic combination comparing parental PvRBP2b and designs and the associated area under the curve (AUC). PvRBP2b, Plasmodium vivax reticulocyte-binding protein 2b.

Article Snippet: Restriction enzyme cloning was used to clone the synthetic DNA fragments (obtained from Twist Biosciences) of PvRBP2b 169-470 designs into pPROEX HTb vector.

Techniques: Infection, Diagnostic Assay, Binding Assay