Bioinformatic modeling, cloning and expression of AntiVEGF disulfid –rich peptide
چکیده
ntroduction: A variety of key human physiological processes rely on
angiogenesis. Furthermore, this process significantly contributes to tumor
progression, invasion and metastasis. As the strongest inducer of angiogenesis,
VEGF and its receptor are targets of therapeutic research for blocking pathological
angiogenesis. Preventing the interaction between VEGF and VEGFR2 by a peptide
is a promising strategy for the development of novel anti-cancer therapeutics. This
study was conducted with the aim of designing and evaluating the anti-VEGF
peptides using bioinformatics and laboratory methods.
Materials and Methods: The effective amino acid sequences in the interaction of
VEGF-A and VEGFR-2 were investigated using Chimera, SPDBV and PyMOL
software. The VEGF binding sites on the receptor were determined and used as the
basis for peptide design. The binding affinity of the resulting peptides was analyzed
by Hex 8.0.0 and ClusPro software. Then, GROMACS v5.0.6 has been used for
Molecular Dynamics (MD) simulation and assessing the stability of target-ligand
complexes. The gene coding for the selected peptide was cloned and expressed in
E. coli BL21. The bacterial cells were cultured and the expressed recombinant
peptide was purified using Ni-NTA chromatography. The reactivity of the peptide
with VEGF was confirmed using western blotting and ELISA assays. Finally, the
inhibition potency of the peptide on human umbilical vein endothelial cells was
assessed using the MTT assay.
Results: Among the three peptides (PepA, SFTI-1- PepA-L1 and Pep1) derived
from VEGFR2, the Pep1 with the best ELISA result and the highest reactivity with
VEGF was selected for further in vitro analysis. Western blot analysis confirmed
the specific reactivity of the selected peptide with VEGF. The MTT assay revealed