The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli

•The N-terminus of human granulocyte-colony stimulating factor (hG-CSF) contains amino acids whose coding sequences are located in the rare codon table of E. coli.•The effect of rare codons on hG-CSF expression level was evaluated through introducing silent mutations in the 5′-end of the coding sequence.•The presence of rare codons following the ATG start codon increases the rhG-CSF expression in E. coli. Presence of the rare codons resulted from the difference in codon usages among organisms is considered as an obstacle to heterologous gene expression. This is especially important for the expression of the genes with eukaryotic origin in Escherichia coli. The N-terminus of human granulocyte colony stimulating factor (hG-CSF) contains amino acids whose coding sequences belong to the rare codons in E. coli. In this study, the effect of rare codons on hG-CSF expression level was evaluated through introducing silent mutations in the 5′-end of the coding sequence. E. coli BL21 (DE3) was used as an expression host. The constructs with the rare codons at the positions following the ATG initiation site of hG-CSF elevated the expression level up to 53–56% of the total cell proteins. This effect may be explained either by the rare codons effects on the early elongation region to reduce ribosome traffic jams in the rest of transcript or by their impacts on reduction of GC content at the beginning region. Mfold RNA server and prediction of the 5′ mRNA secondary structure showed the less stable mRNA secondary structure is, the more hG-CSF expression level would be. However, the minimum free energy of the secondary structure individually, could not indicate this correlation between all constructs. This finding seems empirically important in designing the synthetic genes for production of the recombinant protein in E. coli.