Total chemical synthesis of biologically active vascular endothelial growth factor

Abstract

Efficient access: the 204-residue covalent-dimer vascular endothelial growth factor with full mitogenic activity was prepared from three unprotected peptide segments by one-pot native chemical ligations. The covalent structure of the synthetic VEGF was confirmed by precise mass measurement, and the three-dimensional structure of the synthetic protein was determined by high-resolution X-ray crystallography.

Figure 1

Total chemical synthesis of VEGF. (A) Target amino acid sequence; (B) Synthetic strategy for the total chemical synthesis of VEGF. One-pot sequential native chemical ligations^[18] from three peptide segments is used to make the 102 amino acid residue polypeptide chain; subsequent folding/disulfide bond formation gives the desired covalent dimer VEGF protein molecule. [R = -CH₂-CH₂-SO₃H]

Figure 2

Analytical LC-MS data for the total chemical synthesis of VEGF. Analytical HPLC profiles (λ = 214 nm) are shown, together with online electrospray ionization mass spectrometry (LC-MS) data (inset) corresponding to each major product. (A) Native chemical ligation between Thz19-Arg49 –αCOSR (2, 15 mg, 3.37 μmol, 1.53 mM) and Cys50̃Asp102̃ COOH (3, 22 mg, 3.66 μmol, 1.66 mM) at t < 2 min. 7 is the intermediate MPAA-exchanged product; (B) Native chemical ligation reaction after 1 h: 4 is the ligation product; (C) Crude reaction mixture after Thz– to Cys– conversion using 60 mM methoxylamine. HCl at pH 4: formation of the desired Cys– product 5 was confirmed by a mass decrease of 12 Da; (D) One-pot native chemical ligation of Gly1-Tyr18 –αCOSR (1, 10.4 mg, 4.39 μmol, 2 mM) and Cys19̃Asp102̃ COOH at t = 0 min; (E) Crude reaction mixture after 2 h at pH = 6.8: 6 is the ligation product Gly1-Asp102-COOH [overall yield (after purification) = 19.3 mg, 1.6 μmol, 48% based on limiting peptide segment 2]. Analytical HPLC was performed using a linear gradient (10̃54%) of buffer B in buffer A over 22 min (buffer A = 0.1% trifluoroacetic acid (TFA) in water; buffer B = 0.08% TFA in acetonitrile) on a C-3 (Agilent), 4.6 °— 150 mm column at 40 °C (flow rate = 1 mL/min). R = -CH2-CH2-SO3H and asterisk (*) corresponds to MPAA.

Figure 3

HPLC and mass spectrometry characterization of synthetic VEGF. (A) Analytical HPLC profile (λ = 214 nm) of the purified unfolded 102 amino acid polypeptide [Gly1-Asp102-COOH]; (B) LC-MS of purified unfolded 102 amino acid polypeptide [Gly1-Asp102-COOH]; observed mass: 11932.2±0.7 Da (average of the four most abundant charge states), calculated mass: 11932.54 Da (average isotope); (C) Analytical HPLC profile (λ = 214 nm) of the purified synthetic VEGF protein. Folded VEGF showed a 2.8 min earlier retention time shift compared to the reduced polypeptide; (D) Direct infusion electrospray ionization MS of the synthetic VEGF protein: observed mass: 23848.7±1 Da (mean of the three most abundant charge states), calculated mass: 23849.1 Da (average isotope composition). A decrease of 15.7 Da from twice the monomer mass confirmed the formation of eight disulfides. Note the narrow distribution of the charge states in the folded VEGF dimer compared with the unfolded monomer. Analytical HPLC was performed using a linear gradient (10̃54%) of buffer B in buffer A over 22 min (buffer A = 0.1% trifluoroacetic acid (TFA) in water; buffer B = 0.08% TFA in acetonitrile) on a C-3 (Agilent), 4.6 ×150 mm column at 40 °C (flow rate = 1 mL/min).

Figure 4

X-ray structure of chemically synthesized VEGF. (A) Cartoon representation of the experimentally determined structure of the synthetic protein molecule; (B) Sigma A-weighted 2Fo-Fc electron density map of the VEGF contoured at 1σ encompassing the intermolecular disulfide bridge between the two monomer units; (C) Superposition of the polypeptide chain backbones from the crystal structure of the chemically synthesized VEGF reported here (red) and the reported crystal structure (PDB 2VPF) of the recombinant VEGF (8-109) monomer (blue) with 96 residues aligned with r.m.s.d. = 0.7 Å.

Figure 5

Human umbilical vein endothelial cell (HUVEC) proliferation assay: ED50 observed for the chemically synthesized VEGF (8-109) is 4.6 ng/mL; typical ED50 range for human VEGF-165 is 1–6 ng/mL.