Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11.
A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His(6)). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His(6) and CFTR peptides or His(6) peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.
Turner
Mark S
MS
Infectious Diseases Program, Faculty of Science, Queensland University of Technology, Brisbane, Australia. ms.turner@qut.edu.au
Hafner
Louise M
LM
Walsh
Terry
T
Giffard
Philip M
PM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006639:Histidine; D006801:Humans; D007778:Lactobacillus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C105995:BspA protein, bacteria; C505032:CFTR protein, human; D000939:Epitopes; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C081277:Rib protein, Streptococcus agalactiae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006639:Histidine