Simple and efficient method for heterologous expression of clostridial proteins

Many clostridial proteins are poorly produced in Escherichia coil. It has b een suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli an d the quantities of the corresponding tRNAs in E. coli are not sufficient t o ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coil genes, ileX, argU, and le uM; in E. coli; these genes encode tRNAs that are rarely used in E. coil (t he tRNAs for the ATA, AGA and CTA codons, respectively). Our data demonstra te that amplification of ileX dramatically increased the level of productio n of most of the clostridial proteins tested, while amplification of argU h ad a moderate effect and amplification of leuW. had no effect. Thus, amplif ication of certain tRNA genes for rare codons in E. coil improves the expre ssion of clostridial genes in E. coli, while amplification of other tRNAs f or rare codons might not be needed for improved expression. We also show th at amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative po sitions of the corresponding codons in the coding sequence. Finally, we des cribe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coil.