Quantitative relationship between the mRNA secondary structure of translational initiation region and the expression level of heterologous protein in Escherichia coli.

Translational efficiency in Escherichia coli is strongly influenced by mRNA secondary structure of translational initiation region (TIR). We have previously reported that the expression of heterologous protein is directly related to the minimal folding free energy (ΔG) of the local secondary structure. However, identifying biologically relevant maximum and minimum levels of expression, or exploring the optimal level between them, is a key to successful optimization of heterologous protein expression. To systematically search a large range of the ΔG of TIR, we now present a quantitative analysis of the relationship between expression level and these ΔGs. The ΔG of TIR in green fluorescent protein is found to be linearly correlated with the fluorescence intensity over a range of tenfold change. The result demonstrates that the increasing ΔG of TIR can enhance the expression level linearly with no threshold or plateau.