A de novo designed signal peptide cleavage cassette functions in vivo

Leader peptidase (Lep) is a membrane-bound enzyme of the Escherichia coli inner membrane that serves to remove signal peptides from exported proteins. Statistical and experimental studies of known signal peptides have defined a short C-terminal region that seems to provide the information for correct by Lep. Based on the patterns of conserved amino acids found in this region, we have designed a signal peptide cleavage cassette. This cassette is processed at the expected site when introduced after an uncleaved signal peptide. Furthermore, processing is blocked in the predicted manner when the (-3, -1)-rule for signal peptide is violated. This suggests that current understanding of the sequence requirements for signal peptide is sufficiently advanced to be used in, e.g. protein engineering applications.