Journal: The Journal of Biological Chemistry
Article Title: Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1
doi: 10.1016/j.jbc.2023.104998
Figure Lengend Snippet: Binding of CTX-Cy5 and CTX-displaying phages to various proposed target proteins and human serum albumin in the cobalt-coated bead test. “His-tag isolation and pulldown” Dynabeads were coated with various His-tagged proteins at 0.16 μM for each. The tested target proteins were MMP-2, NRP1, CLC-3 chloride channel, TIMP-2, MT1-MMP (MMP14), annexin A2 (ANX2), α v β 3 integrin (INT), MMP-9, and human serum albumin (HSA). A , the coated beads were blocked with casein and stained with 1 μM CTX-Cy5. Control beads (CONT) were not incubated with any target protein but blocked with casein and stained. B , representative intensity distribution histograms of beads from an experiment of A . C , the coated beads were blocked with casein, incubated with CTX-displaying phages (5.5 × 10 14 particles/ml), and stained to detect M13 phage binding. Control beads (CONT) were not incubated with any target protein but blocked with casein, and phages were also applied and stained. The bar graphs in A and C represent relative fluorescence intensities calculated from median fluorescence intensities of 10,000 events recorded by the flow cytometer and are presented as mean ± SD of three separate experiments with superimposed scatter plot. ∗ and ∗∗∗ indicate statistically significant difference ( p < 0.05 and p < 0.001) from control (ANOVA followed by Dunnett’s test). D , representative intensity distribution histograms of beads from the experiment of C . Note the very similar patterns of binding of fluorescent-labeled and phage-displayed CTX to various target proteins. CTX, chlorotoxin; MMP-2, matrix metalloproteinase 2; NRP1, neuropilin 1; TIMP-2, tissue inhibitor of metalloproteinase 2.
Article Snippet: The used target proteins were the following (with National Center for Biotechnology Information accession number [segment amino acid sequence]; His-tag position: C- or N-terminal; supplier; and catalog number listed in parentheses): MMP2 (NP_004521.1 [amino acids: 110–660]; N-; ProSpec, catalog no.: ENZ 769), NRP1 (NP_001019799.2 [amino acids: 2–644]; C-; Sino Biological; catalog no.: 10011-H08H); MMP-9 (NP_004985.2 [amino acids: 20–701]; C-; ProSpec; catalog no.: ENZ 1091), TIMP-2 (NP_003246.1 [amino acids: 27–220]; N-; ProSpec; catalog no.: ENZ-646), MMP 14 (NP_004986 [amino acids: 24–524]; C-; ThermoFisher; catalog no.: RP77533), ANX2 (NP_001002857.1 [amino acids: 1–339]; N-; ProSpec; catalog no.: PRO-777), α v β 3 integrin (AAA52589.1 & NP_002196.4 heterodimer; both with C-terminal His tag; Native Antigen Company; catalog no.: REC31719-100), CLC-3 (NP_001820.2 [amino acids: 1–818]; C-; Creative Biomart; custom made by recombinant expression in E. coli ), and human serum albumin (NP_000468.1 [amino acids: 25–609]; C-; Abcam; catalog no.: ab217817).
Techniques: Binding Assay, Isolation, Staining, Control, Incubation, Fluorescence, Flow Cytometry, Labeling
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