The effect of all possible codons corresponding to the second and third amino acid (isoleucine and threonine) on the expression level of hirudin in E. coli has been analysed. These levels could not be correlated with changes in primary and secondary mRNA structure. A decrease in the rate of synthesis and of product accumulation follows the introduction for ile of the ATA codon which is of very low usage, and for thr of the ACC codon, which results in homology of the mRNA with the 3'-end of 16S rRNA. The results are discussed according to current concepts of protein expression in E. coli.
