Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution overview. ( A ) SNAP25 sub-family proteins and their isoforms (CLUSTAL 2.1 ). ( B ) Co-crystal structure (PDB: 1XTG ) of LC/A (white) and SNAP25 (dark gray). Eight substitutions in LC/A drive SNAP23 specificity (teal) through substrate control loops (pink) alongside prior substitutions (light gray). ( C ) Platform for the directed evolution of SNAP23 substrate specificity. 1. Random or site-directed mutagenesis (e.g., site-saturation); 2. QC by DNA sequencing; 3. High-throughput protein production; 4. Measure V 0 23 and V 0 25 for substrate specificity; 5. Confirmation screens. The most specific and consistent variant from the DARET assay entered the next round of directed evolution. ( D ) Sequence alignment of substrates used for screening (UniProt: P60880, O00161). The SNAP binding exosites in LC/A (residue numbers above) and cleavage site (scissors) are shown. The gradient of color indicates homology from identical (white, *), to strongly similar (light gray, :), weakly similar (teal, .), or dissimilar (dark teal, space).

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques: Control, Mutagenesis, DNA Sequencing, High Throughput Screening Assay, Variant Assay, Sequencing, Binding Assay, Residue

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution of a SNAP23-specific LC/A. ( A ) Improvement in specificity index through directed evolution in salt-free buffer. The most specific variant from each round was assayed multiple times (replicates shown). The average specificity index (horizontal bars) and standard deviation (error bars) are shown. The final dilution of cell lysates for screening is indicated by color code. ( B ) Increased specificity index achieved through directed evolution in salt buffer. ( C ) Roadmap for directed evolution showing the fold increase in specificity indices over eight rounds (R1 to R8). As shown in Fig. S1, the substrate specificity of the qmLC/A variant is sensitive to screening dilution; therefore, each point represents the variant’s average specificity normalized to the qmLC/A average specificity at the corresponding screening dilution. Advancement to the next round (solid line) weighed specificity index, protein solubility, and stability.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques: Variant Assay, Standard Deviation, Solubility

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Enzyme kinetics of LC/A variants.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques:

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Characterization of omLC/A cleavage. ( A ) Rates of SNAP cleavage by batch-expressed, purified qmLC/A and ( B ) omLC/A at the indicated DARET substrate concentrations (n = 3). ( C ) Deconvoluted ESI–MS of recombinant, full-length SNAP23 (fl-S23) treated with DTT and iodoacetamide to carbamidomethylate its six cysteines (6 × CAM). The mass spectrum of intact fl-S23 incubated with buffer (top, black) is compared to that of fl-S23 incubated with omLC/A (bottom, teal). Intact fl-S23 (1) and cleaved fl-S23 (cl. fl-S23, 2) peaks are labeled. Additional marked peaks correspond to the masses of peaks 1 and 2 plus one additional CAM (+ 57 Da), likely resulting from overalkylation by iodoacetamide . The cleaved peptide was not directly observed, but inferred from the mass difference of peaks 1 and 2. The deconvolution error is +/- 2 Da. ( D ) Recombinant omBoNT/A was purified by IMAC followed by anion exchange (AEX) chromatography. The omBoNT/A is ≈95% nicked upon DTT reduction as demonstrated by the presence of the HC/A and omLC/A bands. ( E ) In vitro cleavage of recombinant fl-S23 by two independent preparations of omBoNT/A (1 and 2) visualized with a C-terminal anti-SNAP23 antibody; before proteolysis, omBoNT/A was reduced with TCEP. The untreated, wtLC/A, and wtLC/E lanes provide negative controls. ( F ) In cellulo cleavage of SNAP23 and SNAP25 in SiMA-H1 cells infected with adenovirus delivering DNA encoding mCherry/SNAP23. Cells were treated with omBoNT/A or wtBoNT/A or without toxin (ct). Proteins, full-length (fl) or cleaved (cl), were identified by Western blotting with anti-SNAP23, -SNAP25, or -mCherry antibodies (M, MW marker). Full-length images of these gels with multiple exposures where necessary are shown in Fig. S12 and S13).

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques: Purification, Recombinant, Incubation, Labeling, Chromatography, In Vitro, Infection, Western Blot, Marker

Journal: Advanced Science

Article Title: Delivering Antisense Oligonucleotides across the Blood‐Brain Barrier by Tumor Cell‐Derived Small Apoptotic Bodies

doi: 10.1002/advs.202004929

Figure Lengend Snippet: sCABs are transcytosed by BMECs to cross the BBB. a) TEM images showing a phagosome containing a sCAB in the BMEC of a mouse 10 min after an i.v. sCABs injection. The right panel shows a magnified view of the phagophore selected by the red square and arrow. Scale bar: 500 nm. b) TEM images showing the structure of TJs after sCABs passed through microvessels 15 min after an i.v. sCABs injection. The TJs in the red squares are shown at high magnifications on the right. The red arrow depicts sCABs that passed through BMECs. Scale bar: 500 nm. c) Representative fluorescence images showing the intact structure of TJs after sCABs treatment containing Cy5‐ASO. The right panels show magnified views of the area selected by the white square. Scale bar: 25 µm. d) Representative fluorescence microscopy images showing sCABs containing Cy5‐ASO phagocytized by b. End3 cells in the BBB model. Scale bar: 25 µm. e) Representative fluorescence images showing Cy5‐ASO delivered by sCABs but not the naked one was phagocytized by microglial cells in the BBB model 24 h after the incubation. The morphology of microglial cells as imaged with differentia linterference contrast (DIC). Scale bar: 25 µm. f) Fluorescence images showing GFP‐labeled sCABs containing Cy5‐ASO phagocytized by microglia 24 h after the incubation. The right panels show magnifications of the area selected by the white square. Scale bar: 10 µm. g–k) Representative fluorescence images showing the colocalization of sCABs containing Cy5‐ASO with special protein markers ((g) labeled with clathrin, (h) with caveolin‐1, (i) with EEA‐1, (h) with Rab11 involved in endocytosis, and (k) with Snap23). The colocalization of clathrin and caveolin‐1 was examined at 5 min after sCABs incubation, EEA‐1 and Rab11 at 10 min, and Snap23 at 20 min. The right panels show magnifications of the areas selected by the white square. Scale bar: 25 µm. Images are representative of three independent experiments.

Article Snippet: Moreover, Snap23 (anti‐Snap23 antibody, BA2805, Boster, China), a membrane receptor involved in the interaction of endosomes with the basolateral membrane, was examined at 20 min.

Techniques: Injection, Fluorescence, Microscopy, Incubation, Labeling