LPXTGtagged

LPXTGtagged refers to proteins that include the LPXTG sorting motif near their C-terminus, a sequence used by certain Gram-positive bacteria to covalently anchor proteins to the cell wall. Proteins designed this way are typically exported across the membrane and carry a cell wall sorting signal consisting of a hydrophobic region and a positively charged tail, which together direct the protein to the cell surface. The presence of the LPXTG motif generally indicates that the protein can be processed by a sortase enzyme and covalently linked to the peptidoglycan layer.

Mechanism and anchoring are mediated by sortase enzymes, most commonly sortase A in Staphylococcus aureus and related enzymes in other species. The enzyme recognizes the LPXTG motif, cleaves between the threonine and glycine residues, and then catalyzes the formation of a covalent bond between the protein and the amino group of the cell wall cross-bridge. This results in stable, surface-anchored proteins that can display functional domains extracellularly. The X in LPXTG is variable, allowing some flexibility in engineering LPXTG-tagged constructs.

Applications of LPXTG-tagged proteins include surface display of antigens or binding domains for vaccines, diagnostics, and biocatalysis, as well as the presentation of fusion proteins for screening and library construction in Gram-positive bacterial systems. Detection and verification of surface display are commonly performed by flow cytometry, immunofluorescence, or western blotting of cell wall extracts.

Limitations and considerations include dependence on functional sortase activity, the need for proper signal peptides and CWSS, potential effects on cell physiology, and the fact that this strategy is largely restricted to Gram-positive bacteria. Researchers employing LPXTG-tagged constructs must optimize tag position, expression levels, and host strain to achieve reliable surface display.