Journal: Current protocols in protein science

Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

doi: 10.1002/cpps.38

Figure Lengend Snippet: Sortase-catalyzed transpeptidation. Wild-type and engineered variants of sortase A from Staphylococcus aureus recognize substrates containing an LPXTG motif. The active site cysteine of sortase A cleaves between the threonine and glycine residues to generate a thioester-linked acyl enzyme intermediate. This intermediate is then attacked by an oligoglycine nucleophile, which releases the sortase enzyme and generates a site-specific ligation product linked via a native amide bond. This reaction can be harnessed to generate proteins site-specifically labeled at the N- or C-terminus.

Article Snippet: Protocols for sortase expression are also included here, and expression plasmids for both enzymes are conveniently available through the online gene repository Addgene, or via request from the authors. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 15.3.1 caption a7 Sortase-catalyzed transpeptidation.

Techniques: Ligation, Labeling

Journal: Current protocols in protein science

Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

doi: 10.1002/cpps.38

Figure Lengend Snippet: Site-specific labeling of target proteins using sortase-catalyzed transpeptidation. (A) Reaction scheme for installing modifications at the protein C-terminus. Target proteins contain the requisite sortase substrate motif (e.g., LPETGG) separated from the body of the protein with an optional GGGGS linker. The sortase cleavage site is followed by an optional tag, such as His6, to assist with reaction monitoring and labeled protein purification. For C-terminal labeling, the target protein is paired with an oligoglycine nucleophile tethered to the desired modification. (B) Reaction scheme for installing modifications at the protein N-terminus. Target proteins serve as the reaction nucleophile, and contain one or more N-terminal glycines. Protein targets are paired with synthetic peptides containing the sortase recognition site (e.g., LPETGG) and the modification of interest. (C) Representative examples of peptide probes compatible with sortase-mediated C-terminal (left) or N-terminal (right) labeling.

Article Snippet: Protocols for sortase expression are also included here, and expression plasmids for both enzymes are conveniently available through the online gene repository Addgene, or via request from the authors. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 15.3.1 caption a7 Sortase-catalyzed transpeptidation.

Techniques: Labeling, Protein Purification, Modification