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For many proteins, the N- or the C-terminus make essential contributions to substrate binding, for protein-protein interactions, or for anchoring the proteins to a membrane. In other circumstances, at least one of the termini is buried within the protein, rendering it inaccessible to labeling. The possibility of selective modification of one of the protein’s termini may present unique opportunities for biochemical and biological applications. We describe sortase-mediated reactions to selectively label the N-terminus of a protein with a variety of functional groups. If sortase, the protein of interest, and a suitably functionalized label are available, the reactions usually require less than 3 hours.
Modification of proteins with fluorophores or other compounds of interest enables creation of novel biological tools to study cellular pathways and molecular mechanisms 1,2. Conjugation of a toxic moiety or antigen to a targeting antibody expands the use of these proteins for cellular delivery purposes, while reducing toxic side effects 3–5. The key to tagging the protein of interest without disrupting its structure or function is selective site-specific labeling. Specific labeling at the N-terminus of a protein is often the only option available, either because of the constraints imposed by the protein’s topology 6–8, or because the native C-terminus is essential for function (e.g.,ubiquitin) 9,10 and/or cellular membrane anchoring 11,12 ,which renders cytosolic portions of proteins inaccessible to added sortase if labeling is to be conducted on intact cells.
Maleimide and NHS-ester derived probes are commonly used to modify proteins, as they are reactive with thiol and amino-groups of cysteine and lysine, respectively 13–15. While cysteines and lysines can be introduced at the N-terminus of a protein, the use of side chain-reactive probes lacks selectivity and may also compromise the active site of the protein being labeled. Genetic engineering approaches allow site-specific modification, but may interfere with protein structure 16,17. While the sortase-mediated labeling method described here overcomes many of these challenges, the possibility of unintended alterations that interfere with protein function should always be considered in design and interpretation.
Sortases are expressed by Gram-positive bacteria. They are essential in cell wall biosynthesis 18–21 and covalent attachment of proteins to the peptidoglycan cell wall. Additional background on sortase enzymes and a detailed protocol for C-terminal labeling using sortases can be found in (this issue of) Nature Protocols [ref]. In the specific instance of N-terminal labeling described here, the protein to be labeled is engineered with an exposed stretch of glycines or alanines at its N-terminus when using sortase A from Staphylococcus aureus or Streptococcus pyogenes, respectively. A peptide decorated with a functional group of choice (fluorophores, biotin, lipids, nucleic acids, carbohydrates, etc) and comprising a sortaserecognition motif LPXTG/A sequence (X being any amino acid 22–29) at its C-terminus is then added to the reaction together with sortase. Sortase A cleaves between the Thr and Gly/Ala residues, forming a thioester intermediate with the peptide probe. Nucleophilic attack by the Nterminally modified protein of interest resolves the intermediate, resulting in the formation of a covalent bond between the peptide probe and the N-terminus of the protein (Fig.1). Alternatively, depsi-peptides can be used for N-terminal labeling 29,30. Depsi-peptides feature an ester linkage between the threonine and glycine, instead of an amide peptide bond to yield a more effective leaving group. By using depsi-peptides, the probe concentration in the reaction can be lowered while maintaining yields.
Proteins to be labeled at the N-terminus must display glycine (SrtA, S. aureus) or alanine (SrtA, S. pyogenes) residues at their N-terminus 8,23,29,31. Using standard molecular cloning methods, a stretch of one to five glycines or two to five alanines is introduced, immediately following the initial methionine, which is often removed by methionylaminopeptidase 32. The requisite number of glycines/alanines should be determined empirically, as it depends on the exposure of the N-terminus, although usually three residues suffice. A linker may be interposed between the N-terminal glycines/alanines and the remainder of the protein to improve accessibility 8.
In those cases where the initial methionine is not (completely) removed after protein synthesis, we use an alternative strategy to expose glycine residues at the N-terminus: a thrombin cleavage site (Leu-Val-Pro-Arg-Gly) is inserted to precede the glycine stretch 31,33. Thrombin cleaves between the Arg and Gly residues, thus ensuring that upon cleavage these glycines are exposed on the protein molecule to be labeled. Critical Step: Although thrombin is somewhat specific, we recommend that before choosing this option, the user confirm that the protein of interest is not itself directly susceptible to thrombin cleavage.
Many core facilities devoted to peptide synthesis can deliver modified peptides for use in N-terminal sortase-catalyzed labeling. Alternatively, commercial providers are a readily accessible source of these materials. However, manual synthesis is cost effective, expands the range of modifications possible for the individual user, and for those reasons it is included in this protocol. The following section provides a protocol for the synthesis of 5(6)-TAMRA, biotin labeled, and NHS ester linked probes for sortase A-mediated reactions. Attachment of fluorophores allows microscopy of internalized labeled proteins, while biotin attachment is especially useful for tagging proteins for pull-down experiments with streptavidin beads. For N-terminal reactions, the probe is linked to the N-terminus of a LPETGG peptide for S. aureus and a LPETAA peptide for S. pyogenes sortase A. Although any amino acid can be placed between the proline and threonine, we prefer glutamic acid or other polar amino acids to aid in precipitation of crude peptide after cleavage from the solid phase resin. To reduce the time required for synthesis and purification, Fmoc-Lys(biotin)-OH, Fmoc-Lys(5-TAMRA)-OH, and other pre-conjugated building blocks can be obtained commercially. These building blocks should be coupled to the leucine residue of the sortase recognition sequence.
Kaiser Test TIMING 5 min
Monitor peptide couplings by performing a Kaiser test 34.
Note: This test works on primary amines and does not work for testing the attachment of an amino acid to a Pro residue. Alternative methods such as the acetaldehyde/p-chloroanil test or microcleavage can be used to monitor these reactions 35,36.
Microcleavage Test TIMING 45 min
A) TAMRA-LPETGG Probe
Note: Use Fmoc-Ala-OH in place of Fmoc-Gly-OH to make probes for S. pyogenes sortase A
Resin Preparation TIMING 15 min
Deprotection TIMING 30 min
Coupling Reaction TIMING 2–3 h until pause point, 3.5 h per coupling cycle
PAUSE POINT: The resin can be stored at 4 °C after drying under vacuum. CRITICAL STEP: At this stage, store peptides in their Fmoc-protected form.
Cleavage from Resin TIMING 3 h
Pause point: The crude peptide can be stored as a solid at −20 °C.
Critical step: Verify the identity and purity by LC/MS analysis (linear gradient 5→45% LC/MS buffer B over 10 min). If LC/MS shows that the crude peptide is of sufficient purity, the next steps (12–14) may be omitted and the peptide may be used directly in sortase reactions.
Critical step: Verify the identity and purity by LC/MS analysis (linear gradient 5→45% LC/MS buffer B over 10 min) and NMR spectroscopy.
Pause point: The lyophilized peptide can be stored at −20 °C indefinitely.
B) Biotin-LPETGG Probe
Critical step: Verify the identity and purity by LC/MS analysis (linear gradient 5→45% B in 10 min) and NMR spectroscopy.
Pause point: The lyophilized peptide can be stored at −20 °C indefinitely.
C) Other LPETGG Probes
For the addition of other functional groups or acid-labile substituents, we recommend using probes conjugated via NHS ester couplings.
The protocol for expression and purification of the various sortase A is found (elsewhere) in (this issue of) Nature Protocols.
A) Setting up the reaction conditions TIMING 8 h
PAUSE POINT: The 1 µl aliquots can be frozen at −20 °C until further analysis.
Note: Some products and starting material proteins have similar molecular weights. A gradient gel of appropriate size and porosity may achieve better separation.
Anticipated result: A successful sortase reaction results in the formation of the acyl-enzyme intermediate. Because the concentration of the protein to be labeled is never sufficiently high to resolve all covalent sortase-peptide intermediates, the sortase-peptide intermediate will be detected in all the reactions. Also, the acyl-enzyme intermediate is rather resistant to reducing and denaturing conditions. Thus, one can detect sortase-peptide adducts by fluorescent scanning or western-blot if the LPETG/A peptide contains a tag (e.g., dye or biotin). A sortase reaction often yields a reaction product with a distinct mobility from that of the input substrate and the hydrolysis product. The ability to distinguish the various intermediates critically depends on the MW of the anticipated products and on the gel systems used to analyze them. In cases where the MW difference is too small to be detected by gel, LC/MS analysis can be used to monitor the reaction and provide an estimated yield.
|No or low protein labeling||Not enough LPXTG/LPXTA probe added to the reaction||Reaction conditions have to be determined ad-hoc. Increase the amount of probe to 10 mM and/or decrease the amount of protein nucleophile|
|pH of the reaction buffer not compatible with sortase activity||Ensure that the pH of the reaction buffer is appropriate. Check the pH of the stock solutions. Especially the pH of the probe solutions can be low, due to residual traces of TFA. Lyophilize the probe solution multiple times with water to remove TFA. Neutralize the probe solution with aq NaHCO3 or by adding additional buffer if the pH remains too low|
|Proteolysis of sortase||Verify the integrity of sortase upon reaction by Coomassie staining and/or anti-His blot. The amount of sortase before and after reaction should be equal. If not, consider the presence of a contaminating protease, which most probably co-purified with the protein of interest. We recommend further purification of the protein nucleophile.|
|N-terminal is not adequately exposed||Increase the length of the poly glycine/alanine nucleophile. If the initial Met is not removed during protein expression a thrombin cleavage site should be added prior to the nucleophilic Gly/Ala residue.|
|Wrong strain of sortase is used||Ensure that you use sortase A from S. pyogenes for alanine-based nucleophiles and sortase from S. aureus for glycine modified proteins.|
|Sortase is inactive||Test the preparation of sortase using N-terminal modified GFP as the nucleophile. Too high concentration of DMSO in reaction mixture, originating from peptide stock solution.|
|Not enough sortase added to the reaction||Sortase concentration has to be titrated for each substrate to be labeled.|
|Increase the substrate concentration and/or decrease the amount of protein to be labeled|
|Detection of a white fluffy precipitate during the sortase-labeling reaction||Ca2+ precipitate||Do not use phosphate buffers if working with a Ca2+-dependent sortase.|
|Protein precipitates during the labeling reaction||High concentration of protein, especially when attempting protein-protein fusions||Optimize reaction temperature and incubation time. Less protein may be sufficient to achieve the same reaction yield without precipitation.|
|The protein of interest precipitates at high temperatures||Perform the labeling reaction at RT and extend the reaction time. 10% glycerol may also prevent precipitation|
|Product and starting material comigrate on gel||MW of product and starting material are similar||Analyze by LC/MS or use a gradient gel|