Roles of Mature Domain Targeting Signals (MTSs) for Protein Translocation and Secretion in Lactococcus lactis.

Lactococcus lactis is a potential bacterial cell factory to develop delivery systems for vaccines and therapeutic proteins. Much progress has been made in applications using engineered L. lactis against, e.g., inflammatory bowel disease and cervical cancer, but the improvement of secretion and cell anchoring efficacy is still desirable. A double-labeling method based on biarsenical hairpin binding and nickel-polyhistidine affinity was used for visualization of protein trafficking and the quantification of targeted proteins on the cell surface and in the cytoplasm. To investigate the importance of mature domain targeting signals (MTSs), we generated truncated constructs encoding 126, 66, and 26 amino acid residues from the N-terminus of the basic membrane protein A (BmpA) and fused those with the gene for the human papillomavirus serotype 16 (HPV16) E7 oncoprotein. Overexpression of fusion proteins was observed to come at the cost of cell proliferation. L. lactis cells produced and displayed the shortest fusion protein only with difficulty, suggesting that the entire absence of a homologous sequence containing MTSs significantly impedes the export and surface anchoring of fusion proteins. With 40 amino acids following the signal peptide and containing one MTS, effective translocation was possible. Mutations of MTSs towards increased hydrophobicity resulted in increased secreted and surface-displayed fusion protein, suggesting the potential to design rationally improved constructs.