earticle

영어

Expression level of protein is affected by various factors including mRNA stability, codon usage and productive binding between RBS and ribosome. It is well-known that direct interactions between the Shine-Dalgarno (SD) sequence and ribosome directly influence translation initiation, that is, protein expression level. Here, we proved that secondary structures of the 5’ proximal coding regions are also crucial for translational efficiency by exploring how expression levels of RFP (DsRed-monomer) were dependent on mRNA secondary structures in 5’ proximal coding region. Random silent mutation of 5’ proximal coding region (+4~+48), which makes 13 possible silent mutation, has shown various levels of gene expression. Also we found that the gene expression increased when its mRNA secondary structure was destabilized. Among them, we chose four RFP variants which showed the highest gene expression and found that the RFP fluorescence increases more than 10-fold. From these results, we propose that manipulating 5’ proximal coding region is a powerful methodology to optimization of protein expression.This work was supported by BK21 program from the Korean Ministry of Education and Seoul R&BD Program (NT080612, KU080657).