Background: Fibroblast growth factors (FGFs) are involved in angiogenesis, wound healing and embryonic development. However, one of the causes of cancer cell growth in fibroblast-dependent cancers is FGF7 secreted by fibroblasts. Therefore, antibodies against FGF7 can be used for treatment of these types of cancers. Methods: In previous studies, a phage displaying single domain antibody, D53, against human FGF7 has been identified using the phage display technique. In the present study, D53 was produced and purified in its isolated form. ELISA experiment was performed to evaluate the binding of D53 to FGF7. The mode of interaction of D53-FGF7 was explored using docking study and molecular dynamics (MD) simulations. Results: The expression and purification processes were verified using western blotting and SDS-PAGE analyses. ELISA experiment showed that D53 is able to recognize and bind FGF7. Docking study and MD simulations indicated that compared to dummy VH, D53 has more affinity towards FGF7. Conclusion: The findings in the current study can be useful for generation and development of FGF7 inhibitors with potential use in fibroblast-dependent cancers.
Purpose: DOF (DNA-binding with One Finger) proteins are plant-specific transcription factors which mediate numerous biological processes. The purpose of the current study is to report new naturally occurring mutations in the gene encoding for one of the members of DOF proteins named DOF 4.2. Methods: The expression of zinc finger domain of DOF 4.2 (DOF 4.2-ZF) was investigated by first synthesis of cDNA library using different parts of Arabidopsis thaliana plant. Then the coding sequence for zinc finger domain of DOF 4.2 protein was prepared using nested PCR experiment and cloned into pGEX-6P-1 expression vector. Finally, the prepared construct was used for protein expression. Furthermore, molecular dynamics (MD) simulation was carried out to predict DNA binding affinity of DOF 4.2-ZF using AMBER package. Results: For the first time a new variant of DOF 4.2-ZF protein with three mutations was detected. One of the mutations is silent while the other two mutations lead to amino acid replacement (S18G) as well as introduction of a stop codon ultimately resulting in a truncated protein production. In order to investigate whether the truncated form is able to recognize DNA binding motif, MD simulations were carried out and the results showed that the chance of binding of DOF 4.2-ZF protein to cognate DNA in its truncated form is very low. Conclusion: The findings demonstrated that the observed mutations adversely affect the DNA binding ability of the truncated form of DOF4.2 if it is expressed in the mutant variant of A. thaliana used in this study.
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