Journal: bioRxiv

Article Title: Linking sugar sensing to immunity in plants via O-glycosylation

doi: 10.64898/2026.05.04.722566

Figure Lengend Snippet: (A) MKK5-GFP was affinity purified from 7-day-old WT but not spy-4 seedlings using Aleuria Aurantia Lectin (AAL) immobilized on agarose beads. Coomassie Brilliant Blue staining (CBB) of the membrane shows the loading control. (B) Bio-layer interferometry (BLI) assay showing binding kinetics between SPY and MKK4. 6xHis-SUMO-3TPR-SPY was immobilized to anti-His sensors and dipped into indicated concentrations of GST-MKK4 protein. The dissociation constant (K d ) was calculated by steady-state fitting of GST-MKK4 concentration against the response unit. (C) Co-immunoprecipitation shows in vivo interaction between SPY and MKK5. YFP or SPY-YFP was transiently co-expressed with MKK5-myc in N. benthamiana leaves, immunoprecipitated by anti-GFP antibody, and immunoblotted with anti-GFP or anti-myc antibody. (D) in vitro affinity purification (pull-down) assay shows direct interaction between SPY/SEC and MKK4/5. Recombinant GST, GST-MKK4, and GST-MKK5 were incubated with 6xHis-tagged SPY, SEC, and PCK1, affinity-purified by Glutathione-agarose beads, and immunoblotted using anti-His and anti-GST antibodies. (E) Higher energy collisional dissociation (HCD) mass spectrum shows O-fucosylation on the activation loop peptide of MKK4 spanning amino acids 220-242, after in vitro O-fucosylation by SPY.

Article Snippet: In brief, bait proteins (final concentration 20 μg/mL) were immobilized onto anti-His (Gator bio #160009) or anti-GST biosensors (Gator bio, #160042) using a Gator-plus instrument (Gator Bio).

Techniques: Affinity Purification, Staining, Membrane, Control, Binding Assay, Concentration Assay, Immunoprecipitation, In Vivo, In Vitro, Pull Down Assay, Recombinant, Incubation, Activation Assay

Journal: bioRxiv

Article Title: Linking sugar sensing to immunity in plants via O-glycosylation

doi: 10.64898/2026.05.04.722566

Figure Lengend Snippet: (A) Bio-layer interferometry (BLI) showing binding kinetics between SPY and MKK5 . 6xHis-SUMO-3TPR-SPY was immobilized to anti-His sensors and dipped into indicated concentrations of GST-MKK5 proteins. The dissociation constant (K d ) was calculated by steady-state fitting of GST-MKK5 concentration against the response unit. (B) BLI assay showing interaction between O-fucosylated GST-MKK5 protein with AAL. GST-MKK5 was incubated with SPY in vitro, with or without GDP-fucose (GF) and SOFTI, loaded onto anti-GST biosensors, and analyzed for binding to AAL.

Article Snippet: In brief, bait proteins (final concentration 20 μg/mL) were immobilized onto anti-His (Gator bio #160009) or anti-GST biosensors (Gator bio, #160042) using a Gator-plus instrument (Gator Bio).

Techniques: Binding Assay, Concentration Assay, Incubation, In Vitro

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: (A) Schematic of in situ protein production in nanowell slide and capture-purification on SPOC biosensor chip - (1) A unique gene (plasmid) is printed in each nanoliter volume well, (2) Human cell-free lysate is added to nanowells, for protein expression, (3) Capture slide/biosensor coated with capture ligand is applied on top, to press-seal wells; and incubated, (4) proteins are produced in each well, and are captured on ligand-coated slide/biosensor resulting in pure protein array on biosensor chip (up to 2304 proteins on <1.5 cm 2 sensor surface). (B) Graphical abstract of SPOC SPR approach for deep mutational scanning and screening of therapeutic targets for epitope characterization. Deep mutationally scanned library of the target protein is expressed in nanowells and capture-purified on SPOC biosensor chip using the protein nanofactory system, followed by SPR-based screening of antibody drugs interactions, to generate pico-molar resolution kinetic data simultaneously from thousands of proteins.

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: In Situ, Purification, Plasmid Preparation, Expressing, Incubation, Produced, Protein Array, Biomarker Discovery

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Schematic representation of CD20 showing (A) Primary amino acid sequence of the partial CD20 expressed in this study. Residues involved in rituximab and ocrelizumab binding are indicated. Non-membrane and membrane-spanning residues are unshaded and shaded in gray, respectively. Cysteine residues responsible for disulfide bond formation are highlighted in red. Only the underlined residues were targeted for partial mutational scanning. (B) PyMOL-generated 3D structures of the partial CD20 sequence and fused HaloTag protein as covalently immobilized on SPOC biosensor. The first five residues (TQSFF) and the last nine residues (GIVENEWKR) of the partial CD20 sequence in (a) were not displayed in this PyMol structure. The membrane-spanning region of CD20 is shaded in gray, whereas non-membrane regions are highlighted in white. CD20 PyMol structure was adapted from an online PDB crystal structure (ID: 6VJA). The crystal structure of the SPOC CD20 has not been obtained. Also, the lipid bilayer is shown for visual representation only – CD20 was not displayed on SPOC chip with a lipid bilayer. (C) Orientation of CD20 on the lipid bilayer, highlighting the binding epitopes targeted by rituximab and ocrelizumab (adapted from Delgado et al. ). Residues in the functional epitope regions indicated by the broken box were substituted with alanine, aspartate, lysine, and serine to generate CD20 DMS library.

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: Sequencing, Binding Assay, Membrane, Generated, Functional Assay

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: (A) In silico recombinant plasmid map of CD20 extracellular domain. Each gene variant was subcloned onto a custom cell-free expression-compatible pT7CFE1 plasmid for expression with an N-terminal HaloTag. (B) Graphical depiction of the SPOC chip design used in his study. CD20 ECD domains (gray) were expressed as HaloTag (blue) fusion proteins synthesized cell-free and captured onto SPOC chips. Various amino acid substitutions (colored circles) were introduced within regions of CD20 spanning the epitopes of the therapeutic antibodies, rituximab and ocrelizumab. The SPOC chip was then screened against these antibodies to assess the impacts of epitope mutagenesis on the antibody binding. (C) SPR flowcell image of a SPOC biosensor, showing distinct protein spots as well as a hockey-shaped fiducial (highlighted in red square) for locating each member protein.

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: In Silico, Recombinant, Plasmid Preparation, Variant Assay, Expressing, Synthesized, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Fluorescence assays to validate protein expression from the silicon nanowell slide and capture onto glass slide, and CD20 therapeutic antibody binding. (A) Nanowell slide probed with rabbit anti-Halo immediately after protein expression on the protein nano-factory unit. Prior to expression for capture on SPOC SPR sensors, SPR capture chips were aligned with each array to ensure precise protein capture onto the chip. Each array corresponds to a single SPOC chip. (B) Validation of CD20 therapeutic Ab binding to the CD20 library. The four arrays on a whole-glass capture slide were divided into incubation chambers, with each pair of chambers probed using either rituximab (1:100 dilution in 5% milk PBST) or ocrelizumab (1:50 dilution in 5% milk PBST). Binding was detected using an anti-human IgG Cy3 secondary antibody. In this assay, only CD20 mutant proteins showed detectable binding, while control proteins remained undetected — except for the human IgG scFv control (indicated by white arrows), which bound the secondary antibody as expected. (C) After thAbs binding assay, the glass slide was incubated with rabbit anti-Halo, followed by secondary anti-rabbit alexafluor647 to validate capture. In this assay, all proteins on the array, including the control proteins and CD20 mutant library were successfully expressed and captured, except for T159K and I162S, which showed poor expression levels (indicated by green arrows in the zoomed-in image). This observation is consistent with the capture response heatmap from the SPR assay.

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: Fluorescence, Expressing, Binding Assay, Biomarker Discovery, Incubation, Mutagenesis, Control, SPR Assay

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Heat map of protein expression and capture validation, showing the mean binding response levels of SPOC biosensor proteins to 133 nM mouse anti-HaloTag. Columns show the native amino acid residues of CD20 targeted for substitution. Rows indicate the type of amino acid side-chain substitution. This heat map compares the capture levels of the CD20 mutant library with the wildtype and few control proteins, and validates the presence of CD20 mutants on the chip. Protein capture level was also validated using a mouse anti-HaloTag and anti-p53 antibodies (see for all sensorgrams). Overall, the capture validation result indicates that all CD20 mutants were relatively well-expressed when compared to the wildtype CD20 and controls except variants T159K and I162S, which produced comparatively lower RU values. Therefore, these mutants were ignored in subsequent binding kinetics computation. Crossed cell indicate no substitution for that wildtype residue.

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: Expressing, Biomarker Discovery, Binding Assay, Mutagenesis, Control, Produced, Residue

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Sensorgrams of capture validation of each CD20 mutant protein and some controls on the SPOC protein chip using mouse anti-HaloTag antibody only

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: Biomarker Discovery, Mutagenesis

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Schematic of SPOC platform application in antibody discovery pipelines. Initial antibody library screening (Phases 1.1 & 1.2) is accomplished by cloning antibody sequences in scFv or VHH format as HaloTag fusion constructs for cell-free expression and downstream SPOC chip capture (Chip A). In Phase 1.1, this diverse library of clones is screened with the desired target as analyte (gray) for antibody ligands that bind productively (dotted red circles) and with desirable interaction kinetics. In Phase 1.2, Chip A can be re-screened with target once-again, yet this time pre-bound with either a partner protein or competitive antibody that blocks the desired epitope. In this manner, competitive inhibition of binding to antibody ligands on the biosensor chip can be assessed as a means of functional characterization and epitope clustering. In Phase 2, a second chip is designed taking top clones from Phase 1 and performing deep-mutation scanning (DMS) of the paratopes (Chip B). The target can be screened and paratope substitutions that further tune binding identified. Finally, in Phase 3, a third chip is manufactured where the target itself is expressed and captured as a HaloTag fusion containing various amino-acid substitutions by DMS (Chip C). Top clones identified from Phase 2 can then be screened, and clones with desired epitope engagement (e.g. specificity to certain mutations, or resistance to certain epitope substitutions) can be selected as top lead candidates for further engineering and functional screening in cell-based or in vivo experimentation (e.g. neutralization assays).

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: Library Screening, Cloning, Construct, Expressing, Clone Assay, Inhibition, Binding Assay, Functional Assay, Mutagenesis, In Vivo, Neutralization

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet:

Article Snippet: The SPOC biosensor chip, containing the CD20 mutant library in duplicate, was screened against recombinant antibodies using a custom Carterra SPR instrument.

Techniques: Binding Assay