Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Lactobacillus fermentum BR11 possesses a unique locus encoding repetitive proteins with LPXTG cell wall anchoring signals.
• Two identified proteins, Rlp and Mlp, exhibit similarities to known surface and mucus-binding proteins from other bacterial species.
• The Mlp protein exists in multiple copies within the L. fermentum BR11 genome.

Purpose of the Study:

• To identify and characterize novel cell wall-anchored proteins in Lactobacillus fermentum BR11.
• To evaluate the potential of these proteins, Rlp and Mlp, as vectors for surface display and secretion of heterologous peptides.
• To compare the efficacy of Rlp and Mlp as display systems against the established BspA system.

Main Methods:

• Antiserum against whole L. fermentum BR11 cells was used to identify a locus encoding repetitive proteins.
• Regions of Rlp, Mlp, and BspA were utilized to create fusion proteins for surface display or secretion of peptides (human cystic fibrosis transmembrane regulator protein, His(6) epitope).
• The BspA promoter and secretion signal, along with the Rlp cell wall sorting signal, were employed for covalent anchoring of peptides.
• The Mlp promoter and secretion signal were used to express and export fragments of Mlp fused to peptides.

Main Results:

• Rlp demonstrated significantly better surface display vector performance than BspA, despite lower cellular expression levels.
• Rlp fusions achieved 0.7 mg/L, while BspA fusions reached 4 mg/L, yet Rlp showed superior display efficiency.
• Expression and export of large (328-kDa) and small (27-kDa) Mlp fragments fused to peptides were achieved using the Mlp promoter and secretion signal.

Conclusions:

• The newly identified repetitive proteins Rlp and Mlp from L. fermentum BR11 are promising candidates for protein production and targeting vectors.
• Rlp serves as a highly efficient surface display vector for heterologous peptides.
• Mlp offers a versatile system for expressing and exporting various peptide fragments.