Peptide Modifications: Biotinylation for Peptide-Protein Interactions
The extraordinarily stable, non-covalent interaction between avidin and biotin is one of the most commonly exploited tools in chemistry and biology. Biotin is a component of the vitamin B2 complex. It binds with high affinity to the chicken egg white protein, avidin, and the fungal protein, streptavidin. Avidin and streptavidin are tetrameric proteins that bind four molecules of D-biotin extremely tightly and its binding is the strongest noncovalent interaction known in nature (Kd = 10-15 M).
The interaction between biotin and avidin can be used for protein purification, detection, immobilization, drug targeting, protein structure analysis, and peptide labeling.
The most straightforward method for determining interaction partners of a peptide is to use it as bait in affinity pull-down experiments followed by direct detection of binding proteins. Pull-down assays are useful for both confirming the existence of a protein-protein interaction predicted by other research techniques (e.g., co-immunoprecipitation) and as an initial screening assay for identifying previously unknown protein-protein interactions. The synthetic peptides are usually used in the verification of postulated protein-protein interactions by competitive disruption of binding.
Biotinylated peptides containing a specific function domain and corresponding control unmodified peptides, can be immobilized onto avidin-conjugated beads. The beads are incubated with a sample of interest, such as nuclear extract or purified recombinant protein, and washed to remove unbound proteins. Bound proteins can then be eluted and analyzed by SDS/PAGE and visualized by protein staining. By comparing proteins bound to modified versus unmodified peptides it is possible to identify candidate “reader” proteins for specific function protein.
Biotinylated peptides with specific modifications can be chemically synthesized at >80% purity. Peptides should be approximately 15-20 amino acids in length with the modification of interest in the center of the sequence and at least 6-8 flanking residues on each side. Usually, biotin can be introduced either N-terminally or C-terminally. We recommend N-terminus modification for its higher success rate, shorter turnaround time, and ease of operation. In contrast, the C-terminus modification requires an additional Lysine.
Pull-down Protocol of Biotin-labeled Peptide
Biotinylated peptides are conjugated to avidin beads to generate the resin used for the peptide pulldown. Immobilized streptavidin beads (Pierce) were loaded with biotinylated peptide prior to incubation with cell lysates. Cells were lysed in 1% (v/v) Nonidet P-40, 150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, protease inhibitors (Complete Tablets, Roche Applied Science), and 1 mM sodium orthovanadate as phosphatase inhibitor. Equal amount of protein was incubated with the respective immobilized peptides at 4 °C for 6 h. After extensive washes, bound proteins were eluted from the immobilized peptides by boiling in SDS sample buffer. Alternatively, the bait peptide with its bound proteins was cleaved off the beads by using 50 mM dithiothreitol. Eluates from the active and control bait peptide pull-downs were combined for further analysis.
Sketch for streptavidin-biotin bond-force measurements. Because of their exceptionally high binding affinity, two of the most prominent ligand-receptor pairs are streptavidin-biotin and avidin-biotin. Both proteins have a tetrameric structure, so they can bind up to four ligands.
Peptides corresponding to regions potentially involved in interactions are synthesized. Proteins bind to the bait peptides are eluted and mixed prior to analysis. After digestion of eluted proteins with trypsin, the eluates can be detected by mass spectrometry or SDS-PAGE. The technical is especially useful for the metabolically labeled cells. The following reference described a method using C13 labeled cells. The phosphorylated peptides and unphosphorylated peptides were synthesized separately. The metabolically labeled proteins were incubated with two related forms. The final results identified a larger peak intensity of the C13-labeled form from the phosphorylated bait. This technique present a novel proteomic screen to find direct protein interaction partners using synthetic modified and unmodified peptides. This peptide-protein interaction screen method is specific and reproducible. The unbiased method only requires the binding partner in the cell lysate. The results are also discriminating because a limited number of proteins were retrieved from a large number of proteins in total cell lysates.
Reference: A Novel Proteomic Screen for Peptide-Protein Interactions, J. Biol. Chem. 2004, 279:10756-10764, doi: 10.1074/jbc.M309909200
Background and Introduction
LifeTein routinely synthesize biotinylated peptides for protein-protein interaction studies. Usually, biotin can be introduced either N-terminally or C-terminally (via lysine). We recommend N-terminus modification for its low cost, higher success rate, shorter turnaround time, and ease of operation. Peptides are synthesized from the C-terminus to the N-terminus. N-terminus modification is the last step in the SPPS protocol. No more specific coupling steps are required. In contrast, the C-terminus modification requires additional steps and is usually more complex. However, in principle biotinylation can be positioned anywhere.
Biotin can be separated from the peptide by a variety of different linkers or spacers. It is recommended that a flexible spacer such as Ahx (a 6 carbon linker) be included to render the biotin label more stable or flexible.
- LifeTein offers biotinylation at N or C terminus: Biotin (N terminus), Lys(Biotin)(middle), Lys(Biotin)(C terminus).
- Biotinylation with Ahx linker (or Long Carbon (LC) linker): Biotin-Ahx (N terminus), Lys(Ahx-Biotin)(middle), Lys(Ahx-Biotin)(C terminus).
Protein Research Applications
Peptides that are biotin-labeled are routinely detected or purified with avidin conjugates in many protein research applications: Western blot, enzyme-linked immunosorbant assay (ELISA), immunoprecipitation (IP), peptide affinity purification, immunohistochemistry (IHC), cell surface labeling and flow cytometry/fluorescence-activated cell sorting (FACS).
- Localize surface proteins or other protein interaction complex using a biotinylated peptide as the bait to capture a putative binding partner from a sample. One strategy is using a single-step streptavidin capture of in vivo biotinylated proteins and tandem affinity purification involving one traditional affinity tag (FLAG or 6HIS) coupled with a biotinylation peptide.
- Immobilize biotinylated peptides to affinity support materials such as avidin agarose.
- Incubate crude samples with the solid support.
- Wash away nonbound sample components from the solid support.
- Elute the target molecule together with its associated proteins by altering the buffer conditions.
- The high biotin–streptavidin affinity allows very high stringency washing conditions. This advantage will reduce the background binding that may be observed with other affinity tags or native antibodies. So it is very convenient to determine the ability of two purified proteins to interact (bind) in various buffer conditions.
- There are only a few naturally biotinylated proteins. So the chance for cross-reaction is very low. Biotinylation can thus be used to extract protein-protein interaction information from transcription-translation lysates.
Biotinylated peptides can be used for ELISA or blotting to detect with streptavidin-HRP or affinity-purifying receptor or antibody. The N terminal amine or amine on lysine are needed for the binding reactions. The biotinylated peptides can be tightly immobilized on streptavidin column.
Please click here to get a peptide synthesis service quote now!