Bacterial proteins destined for export from the cytoplasm by the Sec pathway contain an N-terminal signal peptide. This signal peptide is cleaved by signal peptidase upon translocation. Residues in the signal peptide and mature region adjacent to the signal peptidase cleavage site are important for secretion efficiency. Recently we showed that the presence of aromatic amino acids at the second position after the signal peptidase cleavage site (P2’) in Escherichia coli lead to accumulation of precursor protein. This precursor protein still contains the signal peptide, indicating inefficient processing by signal peptidase, LepB. Here we further investigate this bias, focusing on a protein found in Bacillus, TasA, which contains a phenylalanine at P2’. TasA is usually cleaved by a non-essential signal peptidase, SipW, which contains a serine-histidine active site. TasA and SipW are present on the same operon and are expressed during sporulation and biofilm formation. The fusion of the TasA signal peptide, including the early mature region, to maltose binding protein is toxic when overexpressed. Stepwise mutations back to maltose binding protein from TasA enables us to understand which residues in the TasA signal peptide causes this toxic effect when expressed in E. coli. In addition, a similar fusion protein of TasA to beta-lactamase up to P2’ was created. This protein showed a decrease in MIC against ampicillin, as well as only precursor protein visualised by western blot. To study which amino acids are permitted, mutational PCR at each position was done before being transformed and grown on plates containing ampicillin at four times the MIC of TasBla P2’. This analysis showed which amino acids allowed for growth, and to what extent LepB processing had been restored. These studies have allowed us to deepen the understanding on which amino acids at certain positions surrounding the LepB cleavage site are important for processing.