Crucial role of non-hydrophobic residues in H-region signal peptide on secretory production of l-asparaginase II in Escherichia coli.

Protein secretion into the periplasmic space requires several interactions between the amino-terminal signal sequence of the protein and secretion machinery components. Therefore, modification of the components of amino-terminal sequence can be used as a powerful strategy to improve secretion efficiency. The hydrophobic region is an important domain for signal peptide function due to interaction with different components of the secretion apparatus. In this study, to evaluate the effect of hydrophobicity level and secondary structure of the h-domain signal peptide on the secretion efficiency, a series of missense mutations were constructed in the hydrophobic domain of the l-asparaginase II signal peptide. The h-region hydrophobicity level of mutants G8L, T16L, and G8L/T16L was increased compared with the wild-type. In addition, the amino acid glycine as a helix-breaker residue was substituted with leucine (G8L), forming a stable and extended α-helix structure in the h-domain. The effect of introducing an aromatic residue in this region was also investigated by mutant G8F. Our mutagenesis studies showed that increasing the hydrophobicity levels, extending the α-helical conformation, and the introduction of an aromatic residue within the h-region signal sequence reduced the secretion level of asparaginase. These results imply a vital role of non-hydrophobic residues in the H-region.