Protein Ligation in Living Cells Using Sortase

Abstract

Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca2+-independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal sortase-recognition motif (LPXTG); we used proteins with an N-terminal (oligo)glycine as nucleophiles. We show that sortase-dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein-based nucleophiles in different intracellular compartments.

Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease

“…RBCs Carrying Ins [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] Confer Protection Against T1D. We investigated whether this strategy could also be applied to T1D in the NOD/ ShiltJ mouse model.…”

“…generate red cells that have variable numbers of sortaggable proteins on their surface (12,13). Because tolerogenic doses vary among different antigens, it is important to have a source of RBCs that can be modified consistently and reproducibly with a known quantity of antigen.…”

“…Mice are considered diabetic when their plasma glucose levels rise to >250 mg/dL. Insulin B-chain peptide 9-23 (Ins [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] ) is an immunodominant self-antigen, the recognition of which can mediate autoimmune destruction of pancreatic β-cells and impair insulin production and release (21). A single prophylactic transfusion of Kell-LPETGG RBCs sortagged with the Ins 9-23 at 10 wk of age protected NOD/ShiltJ from T1D in 80% of animals until week 30, whereas injection of unmodified RBCs did not (Fig.…”

Sortase-mediated backbone cyclization of proteins and peptides

“…However, this procedure is not yet fully developed. Although expression of a cyclic product in S. cerevisiae proceeded smoothly, expression in human cells led to a 50% yield (Strijbis et al, 2012). In the latter case, recombinant production of a cyclized product would make a subsequent purification step necessary.…”

“…The potential clinical use of cyclic proteins has prompted research to their direct synthesis in cells by recombinant co-expression of a suited linear precursor and sortase in the same cell. Since the Ca 2+ -dependent S. aureus sortase A is not functional intra-cellularly, the Ca 2+ -independent Streptocuccus pyogenes sortase A was used for this purpose (Strijbis et al, 2012;Zhang et al, 2013;Strijbis and Ploegh, 2014). In a proof-of-principle experiment, it was shown that co-expression of S. pyogenes sortase A and GG-GFP-LPETG-Myc (control epitope tag) resulted in efficient backbone cyclization in the cytosol and endoplasmic reticulum lumen of transfected Saccharomyces cerevisiae and human HEK293T cells (Strijbis et al, 2012).…”