Protein Ligation in Living Cells Using Sortase

Sortase A transpeptidase (SrtA) is an enzyme of Gram-positive bacterial origin involved in covalent attachment of proteins to the bacterial cell wall. The enzyme has also been used in a number of protein-engineering applications (reviewed in 1). Sortases recognize substrate proteins bearing a sortase motif, LPXTG for Staphylococcus aureus SrtA and LPXTG/LPXTAA for Streptococcus pyogenes SrtA, but sortases from other Gram-positive bacteria have yet again different recognition motifs (2, 3) and substrate requirements. After recognition of the sortase motif by SrtA, the catalytic cysteine residue in the enzyme's active site serves as a nucleophile to cleave the peptide bond between threonine and glycine (or alanine). Cleavage occurs with concomitant formation of a thioacyl intermediate between substrate and enzyme. This intermediate is then resolved by the N-terminus of an (oligo)glycine nucleophile, thereby creating a new peptide bond that links the substrate to the incoming nucleophile (Figure 1). We refer to this method of protein labeling as sortagging. The method in principle allows protein modifications at the C-terminus, the N-terminus or both (reviewed in 1). Figure 1 Schematic representation of sortase-mediated protein ligation Several areas of application of the sortagging method have emerged, ranging from pure chemistry-based to cell biology and biochemistry. The method has proved versatile especially because the enzyme tolerates a wide variety of substrates in proximity of the cleavage site and in nucleophile modifications 2004, the latter including polyethylene glycol chains 2007, lipids 2008, nucleic acids 2007 and a glycosylphosphatidylinositol anchor analog 2009. Biochemical applications of sortase include the in vitro sortase-catalyzed circularization of proteins such as enhanced green fluorescent protein (eGFP), four-helix bundle cytokine interferon α3 (IFNα3) and granulocyte colony-stimulating factor-3 (GCSF-3), human erythropoietin (EPO) and the wound-healing peptide histatin-1. The resulting circular polypeptides proved to be more stable and at times more active than their linear counterparts (10—12). The reaction conditions for sortase-mediated transacylation are compatible with the environment of live cells. Membrane proteins exposed at the cell surface can serve as substrates for both C- and N-terminal labeling (13—17), even in complete tissue culture medium supplemented with serum components. The site-specific attachment of the nucleophile, the small sizes of nucleophiles that serve as substrates, the reversibility of the reaction and the mild reaction conditions are all attractive features of this labeling method. The mild reaction conditions for the sortase reaction, as well as the ability to execute ligation reactions on the surface of living cells suggested that it might be possible to apply sortase to intracellular protein ligation. We thus set out to adapt the sortagging method for intracellular use. We show that the Ca2+-dependent S. aureus enzyme is not functional intracellularly, but the Ca2+-independent S. pyogenes SrtA is functional in the cytosol and endoplasmic reticulum (ER) lumen of both Saccharomyces cerevisiae and mammalian HEK293T cells. Circularization of proteins in the ER lumen results in secretion of the circular product, opening up the possibility of having recombinant proteins with protein modifications specific for eukaryotes (e.g. N-linked glycosylation) produced and secreted directly as circular products. We also find that GFP-based nucleophiles can be used to sortag multiple protein substrates, both in the cytosol and in the ER.