Double-Labeling Method for Visualization and Quantification of Membrane-Associated Proteins in Lactococcus lactis.

Area of Science:

• Microbiology
• Biotechnology
• Molecular Biology

Background:

• Lactococcus lactis engineered to display recombinant proteins offers potential as a live-cell drug delivery vector for mucosal and oral administration.
• Effective use as a vector necessitates quantitative control of antigens and a thorough understanding of bacterial protein processing mechanisms.

Purpose of the Study:

• To develop a method for simultaneous detection and quantification of proteins on the cell surface and in the cytoplasm of L. lactis.
• To confirm the secretion pathway of membrane-associated proteins in L. lactis.

Main Methods:

• A double-labeling technique using conjugated dyes specific for polyhistidine tags (surface proteins) and tetra-cysteine tags (cytoplasmic proteins).
• A method to block labeling of surface-exposed tetra-cysteine tags to ensure location-specific signals.
• Application of the method to model proteins HtrA and BmpA in L. lactis.

Main Results:

• Simultaneous detection and quantification of targeted proteins on the cell surface and in the cytoplasm were achieved.
• Full-length peptide chains of HtrA and BmpA were detected, confirming their association with the cell membrane via distinct modes.
• The Sec-dependent post-translational pathway was confirmed for the secretion of membrane-associated proteins in L. lactis.

Conclusions:

• The developed double-labeling method provides a convenient tool for monitoring cytoplasmic protein production, export, and surface attachment in L. lactis.
• This method facilitates the optimization of L. lactis for cell surface display applications and live-cell drug delivery.