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

Area of Science:

• Microbiology and Biotechnology
• Protein Engineering
• Vaccine Development

Background:

• Lactococcus lactis is a promising bacterial cell factory for producing vaccines and therapeutic proteins.
• Current applications of engineered L. lactis show potential for treating inflammatory bowel disease and cervical cancer.
• Enhancing protein secretion and cell anchoring efficiency in L. lactis remains a critical area for improvement.

Purpose of the Study:

• To investigate the role of mature domain targeting signals (MTSs) in the secretion and cell surface display of fusion proteins in L. lactis.
• To evaluate the impact of different truncated constructs of the basic membrane protein A (BmpA) fused to the human papillomavirus (HPV) 16 E7 oncoprotein.
• To explore strategies for rationally designing improved fusion protein constructs for enhanced efficacy.

Main Methods:

• Development of a double-labeling method using biarsenical hairpin binding and nickel-polyhistidine affinity for protein visualization and quantification.
• Generation of truncated BmpA constructs (126, 66, and 26 amino acids) fused to the HPV16 E7 oncoprotein.
• Analysis of protein trafficking, cell surface anchoring, and cytoplasmic localization in engineered L. lactis.

Main Results:

• Overexpression of fusion proteins negatively impacted L. lactis cell proliferation.
• The shortest fusion protein (26 amino acids) was difficult for L. lactis to produce and display, indicating MTSs are crucial for export and anchoring.
• Fusion proteins with 40 amino acids and one MTS demonstrated effective translocation; mutations increasing MTS hydrophobicity enhanced secretion and surface display.

Conclusions:

• The presence of MTSs is essential for efficient protein export and surface anchoring in engineered L. lactis.
• Rational design, including optimizing MTS hydrophobicity, can significantly improve the secretion and cell surface display of fusion proteins.
• These findings provide a foundation for developing more effective L. lactis-based delivery systems for vaccines and therapeutics.