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Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

Maximilian Wei‐Lin Popp^1,2, John M. Antos², Hidde L. Ploegh^1,2

¹Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts
²Whitehead Institute for Biomedical Research, Cambridge, Massachusetts

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 15.3

DOI:

10.1002/0471140864.ps1503s56

Online Posting Date:

April, 2009

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Abstract

Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5-aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine peptide and the target protein. By functionalizing the oligoglycine peptide, it is possible to incorporate reporters into target proteins in a site-specific fashion. This reaction is applicable to proteins in solution and on the living cell surface. The method described in this unit only requires incubation of the target protein, which has been engineered to contain a sortase recognition site either at the C terminus or within solvent-accessible loops, with a purified sortase enzyme and a suitably functionalized oligoglycine peptide. Curr. Protoc. Protein Sci. 56:15.3.1-15.3.9. © 2009 by John Wiley & Sons, Inc.

Keywords: sortase; transpeptidation; site-specific labeling; chemoenzymatic labeling

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Introduction
Strategic Planning
Basic Protocol: Site-Specific Protein Labeling Via Sortase-Mediated Transpeptidation
Alternate Protocol: Labeling Cell-Surface Proteins in Living Cells Via Sortase-Mediated Transpeptidation
Support Protocol: Expression and Purification of Sortase A
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: Site-Specific Protein Labeling Via Sortase-Mediated Transpeptidation

Materials

Purified target protein (protein must not be dissolved in phosphate buffer)
Purified sortase A stock solution (see Support Protocol)
100 mM to 500 mM oligoglycine probe stock solution in DMSO or H₂O
Ni-NTA column, optional
10 to 30 mM imidazole, optional
500 mM NaCl, optional
10× sortase buffer: 500 mM Tris×Cl, pH 7.5 (appendix 2E), 1.5 M NaCl, 100 mM CaCl₂

1.5-ml microcentrifuge tubes
37°C water bath

Additional reagents and equipment for SDS-PAGE (unit 10.1), immunoblot (units 10.7 & 10.10), and Coomassie stain (unit 10.5)

Alternate Protocol: Labeling Cell-Surface Proteins in Living Cells Via Sortase-Mediated Transpeptidation

Materials

Target cells
Plasmid encoding target protein
Transfection reagent (Lipofectamine, Invitrogen; Trans IT, Minus; Fugene 6, Roche)
Purified sortase A stock solution (see Support Protocol)
100 mM to 500 mM oligoglycine probe stock solution in DMSO or H₂O
Culture medium (presence of 10% serum does not inhibit the sortase reaction)
Phosphate-buffered saline with 1 mM CaCl₂ and 1 mM MgCl₂ (PBS++)

Plastic culture dishes or chambered coverslips (Lab-Tek II Chambered Coverglass, Nunc)
37°C humidified incubator

Additional reagents and equipment for SDS-PAGE (unit 10.1) and immunoblot (units 10.7 & 10.10)

Support Protocol: Expression and Purification of Sortase A

Materials

Sortase expression plasmid: pQE30 (Qiagen) or pET28a+ (Novagen)
E. coli BL-21(DE3)
Luria Bertani (LB) medium with and without appropriate antibiotic:
- Ampicillin (1000× stock = 100 mg/ml) for pQE30-derived construct
- Kanamycin (1000× stock = 30 mg/ml) for pET28a(+)-derived construct
1 M isopropyl -d-thiogalactopyranoside (IPTG)
Phosphate buffered saline (PBS; appendix 2E)
Lysis buffer: 50 mM Tris×Cl, pH 7.5 (appendix 2E), 150 mM NaCl, 10 mM imidazole, 10% glycerol
10 mg/ml DNaseI; appendix 2E
Ni-NTA agarose slurry (Qiagen)
Elution buffer: 50 mM Tris×Cl, pH 7.5 (appendix 2E), 150 mM NaCl, 500 mM imidazole, 10% glycerol

Culture plates and tubes
37°C incubator with shaking
Spectrophotometer
French press, pre-chilled
1.5 × 12–cm column (Biorad)
Centrifugal spin concentrator with low-molecular-weight (<10 kD) cutoff optional (Millipore)

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Figures

Figure 15.3.1

Site-specific labeling of target proteins by sortase-mediated transpeptidation. (A) Sortase-mediated transpeptidation mechanism. Sortase A recognizes substrates with an LPXTG motif, cleaving the peptide bond between the threonine and glycine (top) and resulting in a thioacyl intermediate (middle). A modified oligoglycine nucleophile then attacks the thioacyl intermediate to yield the transpeptidation product with the probe in amide linkage to the target protein (bottom). (B) Substrate design. Substrate proteins typically have the sortase cleavage site (LPETGG) separated from the body of the protein by an optional Gly₄Ser linker of variable length. Placement of an epitiope tag (His₆, HA-tag, BirA acceptor peptide) C-terminal to the LPETGG motif allows a convenient means of purification and assessing the reaction progress, as this tag is lost upon transpeptidation.

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Figure 15.3.2

Peptide probes compatible with sortase-mediated transpeptidation. (A) General structure of oligoglycine based nucleophiles used in sortase-mediated transpeptidation. (B) Example of a fluorescent probe, consisting of an oligoglycine peptide unit followed by a lysine to which fluorescein is appended.

View Image

Literature Cited

Literature Cited
	Antos, J.M., Miller, G.M., Grotenbreg, G.M., and Ploegh, H.L. 2008. Lipid modification of proteins through sortase-catalyzed transpeptidation. J. Am. Chem. Soc. epub ahead of print.
	Bentley, M.L., Gaweska, H., Kielec, J.M., and McCafferty, D.G. 2007. Engineering the substrate specificity of Staphylococcus aureus sortase A. The beta6/beta7 loop from SrtB confers NPQTN recognition to SrtA. J. Biol. Chem. 282:6571-6581.
	Bentley, M.L., Lamb, E.C., and McCafferty, D.G. 2008. Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus. J. Biol. Chem. 283:14762-14771.
	Boekhorst, J., de Been, M.W., Kleerebezem, M., and Siezen, R.J. 2005. Genome-wide detection and analysis of cell wall-bound proteins with LPxTG-like sorting motifs. J. Bacteriol. 187:4928-4934.
	Chan, L., Cross, H.F., She, J.K., Cavalli, G., Martins, H.F.P., and Neylon, C. 2007. Covalent attachment of proteins to solid supports and surfaces via sortase-mediated ligation. PLoS ONE 211:e1164.
	Chen, I. and Ting, A.Y. 2005. Site-specific labeling of proteins with small molecules in live cells. Curr. Opin. Biotechnol. 161:35-40.
	Chen, I., Howarth, M., Lin, W., and Ting, A.Y. 2005. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 22:99-104.
	Clow, F., Fraser, J.D., and Proft, T. 2008. Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A. Biotechnol. Lett. 309:1603-1607.
	Foley, T.L. and Burkart, M.D. 2007. Site-specific protein modification: Advances and applications. Curr. Opin. Chem. Biol. 111:12-19.
	George, N., Pick, H., Vogel, H., Johnsson, N., and Johnsson, K. 2004. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126:8896-8897.
	Griffin, B.A., Adams, S.R., and Tsien, R.Y. 1998. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269-272.
	Ilangovan, U., Ton-That, H., Iwahara, J., Schneewind, O., and Clubb, R.T. 2001. Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 98:6056-6061.
	Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., and Johnsson, K. 2003. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 211:86-89.
	Lin, C.W. and Ting, A.Y. 2006. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J. Am. Chem. Soc. 128:4542-4543.
	Mao, H., Hart, S.A., Schink, A., and Pollok, B.A. 2004. Sortase-mediated protein ligation: A new method for protein engineering. J. Am. Chem. Soc. 126:2670-2671.
	Marks, K.M., Rosinov, M., and Nolan, G.P. 2004. In vivo targeting of organic calcium sensors via genetically selected peptides. Chem. Biol. 113:347-356.
	Marraffini, L.A., Dedent, A.C., and Schneewind, O. 2006. Sortases and the art of anchoring proteins to the envelopes of Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 701:192-221.
	Miller, L.W., Sable, J., Goelet, P., Sheetz, M.P., and Cornish, V.W. 2004. Methotrexate conjugates: A molecular in vivo protein tag. Angew. Chem. Int. Ed. Engl. 43:1672-1675.
	Parthasarathy, R., Subramanian, S., and Boder, E.T. 2007. Sortase A as a novel molecular “stapler” for sequence-specific protein conjugation. Bioconjug. Chem. 182:469-476.
	Popp, M.W., Antos, J.M., Grotenbreg, G.M., Spooner, E., and Ploegh, H.L. 2007. Sortagging: A versatile method for protein labeling. Nat. Chem. Biol. 311:707-708.
	Popp, M.W., Artavanis-Tsakonas, K., and Ploegh, H.L. 2008. Substrate filtering by the active-site crossover loop in UCHL3 revealed by sortagging and gain-of-function mutations. J. Biol. Chem. epub ahead of print.
	Pritz, S., Wolf, Y., Kraetke, O., Klose, J., Bienert, M., and Beyermann, M. 2007. Synthesis of biologically active peptide nucleic acid-peptide conjugates by sortase-mediated ligation. J. Org. Chem. 72:3909-3912.
	Pritz, S., Kraetke, O., Klose, A., Klose, J., Rothemund, S., Fechner, K., Bienert, M., and Beyermann, M. 2008. Synthesis of protein mimics with nonlinear backbone topology by a combined recombinant, enzymatic, and chemical synthesis strategy. Angew. Chem. Int. Ed. Engl. 47:3642-3645.
	Samantaray, S., Marathe, U., Dasgupta, S., Nandicoori, V.K., and Roy, R.P. 2008. Peptide-sugar ligation catalyzed by transpeptidase sortase: A facile approach to neoglycoconjugate synthesis. J. Am. Chem. Soc. 130:2132-2133.
	Tanaka, T., Yamamoto, T., Tsukiji, S., and Nagamune, T. 2008. Site-specific protein modification on living cells catalyzed by sortase. Chembiochem 95:802-807.
	Ton-That, H., Liu, G., Mazmanian, S.K., Faull, K.F., and Schneewind, O. 1999. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc. Natl. Acad. Sci. U.S.A. 96:12424-12429.