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.

Figure 1. Schematic representation of sortase-mediated protein ligation

A substrate protein containing an LPXTG motif near the C-terminus followed by an epitope tag is cleaved between the threonine and the glycine by sortase, thereby releasing the epitope tag (top). The formed acyl-enzyme intermediate can be resolved by an (oligo)glycine-based nucleophile (middle), resulting in the release of sortase and the formation of a substrate-nucleophile ligation product (bottom). Figure adapted from Dasgupta et al. 2011.

Figure 2. GFP circularization by S. pyogenes SrtA in S. cerevisiae and HEK293T cytosol

A) Galactose induction of S. cerevisiae strains expressing cytosolic circularizable G-eGFP-LPETG-Myc substrate under control of the constitutive CIT1 promoter with S. aureus or S. pyogenes sortase under control of the inducible GAL promoter. Samples were taken after 0, 4 and 8 h of galactose induction. Cells expressing either G-eGFP-LPETG-Myc or S. pyogenes sortase (mix control, first two lanes) were mixed before lysis and prepared for immunoblot like the other samples (mix control, third lane). Fractions marked with asterisks were analyzed by MS. B) MS/MS spectrum of a tryptic fragment of the circular GFP showing the ligation of C-terminal residues (YKLPET) to the N-terminal residues (GTIPL). The linkage peptide was found in the (**) sample, but not in the (*) sample. C) Anti-HA immunofluorescence on HEK293T cells expressing S. pyogenes sortase targeted to cytosol (Cyt-SrtA) or plasma membrane (PM_palm-SrtA). Scale bar represents 7 µm. D) Immunoblot analysis of HEK293T cell expressing different combinations of G-eGFP-LPETG-Myc substrate, Cyt-SrtA and PM_palm-SrtA. Cells expressing either G-eGFP-LPETG-Myc or Cyt-SrtA were mixed before lysis and treated like the other samples (mix control). Experiments were performed multiple times, representative experiment are shown.

Figure 3. Secretion of circular proteins through ER sortagging

A) Scheme showing sortase-mediated secretion of circularized eGFP from the ER. B) Intracellular GFP localization in the indicated S. cerevisiae strains after overnight growth minimal glucose medium. Scale bar represents 5 µm. C) Immunoblot analysis of (SP)-G-eGFP-LPETG-HDEL-Myc substrate modification by soluble ER-SrtA and TM_Sec66-SrtA under control of the GAL promoter. Samples were collected at 0, 4 and 8 h of galactose induction. Total cell pellet lysates were analyzed (top) and anti-GFP immunoprecipitation was performed on the culture media to detect secreted GFP (bottom). Fractions marked with asterisks were analyzed by MS. D) MS/MS spectrum of a tryptic fragment of the circular GFP showing the ligation of C-terminal residues (LPET) to the N-terminal residues (GTIPLGSVSK). The linkage peptide was found in the (**) secreted GFP samples, but not in the (*) pellet or secreted samples. E) Quantification of secreted circular GFP product. A standard 50 mL galactose induction starting with 10 OD units (P, 0 h, ∼350 µg of total protein) yielded about 0.75 µg of circular GFP product (S, 8 h) as compared to purified GFP. Experiments were performed multiple times, representative experiments are shown.

Figure 4. Kinetics of cytosolic sortase reactions with Rac1 substrate and G₅-eGFP nucleophile

A) Immunoblot analysis of HEK293T cells transfected with different combinations of Rac1-LPETG-Myc substrate, G₅-eGFP nucleophile and Cyt-SrtA or PM_palm-SrtA plasmids. Cells expressing Rac1 substrate and G₅-eGFP nucleophile were mixed with cells expressing Cyt-SrtA before lysis and treated like the other samples (mix control). B) Radioactive pulse-chase experiment on HEK293T cells transfected with the indicated Rac1 substrate, GFP nucleophile and sortase constructs. Cells were pulse-labeled for 20 min with [³⁵S]methionine/cysteine and chased for 0, 30, 60 and 120 min. Immunoprecipitations (IP) were performed with anti-GFP, anti-Myc, anti-HA or anti-PDI antibodies. All experiments were performed multiple times, representative experiments are shown.

Figure 5. ESortagging of Kar2 substrate with G₅-GFP nucleophiles in the ER lumen

Galactose induction of S. cerevisiae kar2::Kar2-LPETG-Myc-HDEL strain constitutively expressing (SP)-G₅-eGFP or (SP)-G₅-eGFP-HDEL nucleophile with soluble ER-SrtA or TM_Sec66-SrtA under control of the GAL promoter. Sortase expression was induced by growth on galactose-containing media. Cell pellets were harvested (0 and 6 h fractions) and an anti-GFP immunoprecipitation was performed on culture media of the 6-h time-point to detect secreted GFP (S fractions). The experiment was performed multiple times, a representative experiment is shown.

Figure 6. Sortase-mediated protein ligations using p97 substrate and G₅-eGFP nucleophile

A) Immunoblot analysis of HEK293T cells expressing different combinations of the G-His-p97-LPSTG substrate, G₅-eGFP nucleophile and Cyt-SrtA constructs. Cells expressing either substrate(s) or Cyt-SrtA were mixed before lysis and treated like the other samples (mix control). B) Radioactive pulse-chase experiment on HEK293T cells transfected with the indicated p97 substrate, GFP nucleophile and sortase constructs. Cells were pulse-labeled for 20 min with [³⁵S]methionine/cysteine and chased for 0, 2 and 4 h. Immunoprecipitations (IP) were performed with anti-p97, anti-GFP, anti-HA or anti-PDI antibodies. Arrows indicate the p97-GFP ligation product. Experiments were performed multiple times, representative experiments are shown.