Exploration of Length and Hydrophobicity Requirements for Optimal α-Amylase Secretion in Bacillus subtilis Based on Rational Design.

The secretory (Sec) pathway is the major pathway of Bacillus subtilis for protein export from the cytoplasm to the extracellular milieu. An amino-terminal signal peptide is required to initiate protein translocation across the cytoplasmic membrane via Sec. Generally, Sec pathway signal peptides of B. subtilis are around 20-30 amino acids long and tend to be relatively hydrophobic compared to signal peptides from other organisms. In view of the importance of signal peptides for high-level secretory protein production, understanding how signal peptide structure and length influence the efficiency of protein secretion is crucial. Accordingly, the present study was aimed at investigating the effects of signal peptide size and, in particular the subdomain sizes of a signal peptide, in relation to protein secretion efficiency. To this end, a rational engineering approach was employed for the design of shortened signal peptides that can still drive effective protein secretion. This involved in silico interaction studies with the signal recognition particle, which is involved in protein targeting from the ribosome to the membrane. Our results show that the hydrophobic H-domain of a B. subtilis signal peptide can be shortened from seventeen to seven hydrophobic amino acid residues without a detectable decrease in protein secretion.