Iranian

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A new strategy for construction of synthetic gene encoding human basic fibroblast growth factor comprising DNA annealing-ligation and augmentation by polymerase chain reaction was introduced. The sequence of the gene and corresponding amino acid chain were modified in order to increase stability of the protein. First, 300 bp and 160 bp fragments of the gene were assembled from 18 oligonucleotides and ligated separately. Then, the shorter fragment was completed by using PCR and combined with the longer one in a proper orientation in pUC 19. One extra nucleotide that had been found in the gene after DNA sequencing and resulted in frame shift, was rectified through the use of PCR directed mutagenesis. Finally, 5'-terminal region of the gene was augmented by means of PCR in order to restore the N-terminal part of the protein and to introduce the NdeI recognition site. The gene was subcloned into the inducible pET-3a expression vector under control of T7 promoter and expressed in Escherichia coli. The identity of the recombinant protein and level of expression were detected by using Western blot analysis and immunoassay. The proposed method has provided a useful strategy for synthesizing modified proteins that might be applied for protein engineering.

Keywords: gene synthesis, fibroblast growth factor, polymerase chain reaction, gene expression, recombinant protein

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