Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions

摘要: Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature size biological or chemical targets involved. They require minimal target, underlying reactions completed in a reasonable amount time under physiological conditions. Sortase-mediated transpeptidation meet these criteria are compatible other labeling methods. Here we describe expression purification conditions two sortase A enzymes that have different recognition sequences. We also provide protocol allows functionalization any given protein at its C terminus, or, select proteins, an internal site. The target is engineered sortase-recognition motif (LPXTG) place where desired. Upon recognition, cleaves between threonine glycine residues, facilitating attachment exogenously added oligoglycine peptide modified functional group choice (e.g., fluorophore, biotin, lipid). Expression takes ∼3 d, sortase-mediated take only few minutes, but reaction times can extended increase yields.