Vegf a121

Manufactured by Thermo Fisher Scientific

VEGF-A121 is a recombinant human Vascular Endothelial Growth Factor A isoform. It is a secreted protein that promotes angiogenesis and vascular permeability.

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4 protocols using vegf a121

Procurement and Preparation of Growth Factors

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All GFs and chemokines were purchased in their mature forms, highly pure (>95% pure), carrier-free, and lyophilized^{35 (link)}. VEGF-A121, VEGF-A165, PlGF-1, PlGF-2, PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, FGF-1, FGF-2, FGF-6, FGF-7, FGF-9, FGF-10, FGF-18, BMP-2, BMP-3, BMP-4, BMP-7, β-NGF, NT-3, BDNF, IGF-1, IGF-2, HB-EGF, CXCL-11, and CXCL-12α were purchased from PeproTech. CXCL-12γ was purchased from R&D systems. Except for PDGF-DD and BMP-7, which were produced in eukaryotic cells, all GFs were produced in Escherichia coli and thus were not glycosylated. All GFs were reconstituted and stored according to the provider’s instructions to regain full activity and prevent loss of protein.

Ishihara J., Ishihara A., Fukunaga K., Sasaki K., White M.J., Briquez P.S, & Hubbell J.A. (2018). Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing. Nature Communications, 9, 2163.

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Panning for Target-Specific DutaFabs

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For DutaFab panning, various protein targets were expressed from synthetic DNA and purified, or sourced from commercial suppliers. In this study, targets were purchased from Peprotech (VEGFA-121, catalog number 100-20A; PDGF-BB, catalog number 100-14B). Targets were biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (ThermoFisher catalog number A39258), according to manufacturer’s instructions. For naïve selections, phage library panning was typically performed in 4 rounds, wherein the first round was performed with 100 nM of biotinylated target pre-immobilized on Dynabeads M-280 Streptavidin (Thermofisher catalog number 11206D), and rounds 2–4 were performed with 75 nM, 15 nM, and 3 nM of biotinylated target in solution, followed by capture of Fab-on-phage/target complexes on the M-280 beads. For affinity maturation, all panning rounds were performed with the biotinylated target in solution, using various target concentrations in different selection arms. Captured phage clones bearing target-specific DutaFabs were eluted from the M-280 beads using 100 mM DTT, used for infection of log-phase TG1 E. coli cells, and rescued using M13 K07 helper phage, according to standard protocols.

Beckmann R., Jensen K., Fenn S., Speck J., Krause K., Meier A., Röth M., Fauser S., Kimbung R., Logan D.T., Steegmaier M, & Kettenberger H. (2021). DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously. Nature Communications, 12, 708.

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Quantifying VEGF and Complement Binding

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The binding ability and affinity of VID, CID, or IgG to VEGF-A165, VEGF-A121, PIGF, C3b, or C4b were measured by enzyme-linked immunosorbent assay (ELISA). VEGF-A165, VEGF-A121, PIGF, and IgG were purchased from PeproTech. C3b and C4b were purchased from Millipore. Briefly, 50 ng VEGF-A165, VEGF-A121, and PIGF and 250 ng C3b and C4b in 100 μL PBS were added into 96-well plates and incubated at 4°C overnight. After washing the plate three times with 300 μL PBS, the plates were incubated with blocking solution (1% BSA in 100 μL phosphate-buffered saline [PBS]) at 37°C for 2 h. Serially increasing amounts (0.05–50 nM) of VID or CID in 100 μL blocking solution were added to the plate and incubated at 37°C for 2 h. IgG served as the negative control. After a series of reactions, the reactive colors were analyzed at an optical density of 450 nm (OD₄₅₀) using a microplate reader (MK3, Thermo Fisher Scientific, Waltham, MA, USA)

Wang H., Li Y., Shi G., Wang Y., Lin Y., Wang Q., Zhang Y., Yang Q., Dai L., Cheng L., Su X., Yang Y., Zhang S., Li Z., Li J., Wei Y., Yu D, & Deng H. (2019). A Novel Antitumor Strategy: Simultaneously Inhibiting Angiogenesis and Complement by Targeting VEGFA/PIGF and C3b/C4b. Molecular Therapy Oncolytics, 16, 20-29.

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Crystallization of DutaFab-VEGFA-121 Complex

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To obtain crystals of the DutaFab in complex with VEGFA-121 (Peprotech catalog number 100-20A) both proteins were mixed at a 1:1 molar ratio in relation to VEGF monomer. The complex was concentrated to 11 mg/ml and crystallization was performed by hanging drop vapor diffusion against 0.1 M MES pH 6.5 and 1.6 M magnesium sulfate at 20 °C. Needle-shaped crystals grew in about 120 days and were frozen in liquid nitrogen with 20% glycerol as cryo-protectant.

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