Molecular cloning of human basic fibroblast growth factor gene (hbFgf) and in silico analysis of physico-chemical characteristics of bFGF protein

Document Type : Research Paper

Authors

Department of Biotechnology Engineering, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran

Abstract

Objective
This study aimed to clone the human basic fibroblast growth factor (hbFGF) gene into the pCAMBIA1304 binary vector for recombinant bFGF protein production in plant expression systems.
 
Materials and Methods
The full-length open reading frame (ORF) encoding the hbFGF gene was inserted into the pCAMBIA1304 binary vector and subsequently cloned into Escherichia coli strain DH5α. The recombinant expression vector, designated pCAMBIA-hbFGF, was then introduced into competent Agrobacterium tumefaciens strains EHA105, GV3101, and LBA4404.
 
Results
The hbFGF gene, comprising 561 nucleotides, encoded a protein of 155 amino acid residues. The calculated molecular mass and predicted isoelectric point (pI) of the deduced polypeptide sequence were 17.25 kDa and 9.58, respectively. The instability index, aliphatic index, and hydrophobicity index of the bFGF protein were calculated as 36.60, 68.00, and -0.511, respectively, indicating high stability and a hydrophilic nature. Analysis using ProtScale and TMHMM programs confirmed the hydrophilic properties of bFGF and the absence of transmembrane domains. Additionally, the MotifScan program identified a conserved fibroblast growth factor (FGF) family domain in the hbFGF sequence, while no potential N-glycosylation sites were detected. The predicted secondary structure of the mRNA sequence, calculated using the RNAfold program, exhibited a minimum free energy of approximately -136.90 kcal/mol, indicating relatively high stability with low entropy. The simulated three-dimensional structure of the hbFGF protein revealed a pyramidal arrangement of 10 non-parallel β-sheets. Ramachandran plot analysis confirmed that 86.3% of the amino acid residues were organized in regular structures, primarily non-parallel β-sheets, with only two residues (Thr8 and Gly24) exhibiting unusual ϕ and ψ angles. Phylogenetic analysis demonstrated a high degree of similarity with Pan troglodytes, Pongo abelii, and Camelus dromedarius, showing 100%, 100%, and 98.71% sequence similarity, respectively.
 
Conclusions
The bFGF protein was identified as a stable, hydrophilic protein without potential N-glycosylation sites. These characteristics suggest that the bFGF protein may be successfully produced heterologously in plant expression systems for potential commercialization.

Keywords

References

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Agricultural Biotechnology Journal
Volume 17, Issue 1
March 2025
Pages 1-26

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