The human body is composed of billions of cells, and various complex life activities in the body depend on interactions between biological macromolecules, among which proteins are the executors of life activities and the identification of protein-protein interactions (PPIs) is the key to elucidate the molecular mechanisms of complex cellular physiological processes.
Traditional protein interaction screening techniques including Yeast two hybrid (Y2H) and Affinity purification-Mass spectrometry (AP-MS) have limitations. For example, they are not suitable for studying interactions of membrane proteins, and cannot detect weak, transient and dynamic interactions between proteins in vivo.
In recent years, the powerful Enzyme-Catalyzed Proximity Labeling-based Method has been increasingly applied for identifying protein interactions in live cells. This technique mainly labels neighboring proteins by enzyme-catalyzed covalent modifications and combines with mass spectrometry to identify modified proteins, which largely compensates for the shortcomings of traditional protein-interaction screening techniques. There are four main Enzyme-Catalyzed Proximity Labeling Systems: BioID (Proximity-dependent Biotin Identification), APEX (Engineered Ascorbate Peroxidase), HRP (Horseradish Peroxidase) and PUP-IT (Pupylation-based Interaction Tagging).
BioID (Proximity-dependent Biotin Identification)
BioID) technology is based on the R118G mutant of BirA* (Biotin Ligase). Wild type BirA can efficiently activate D-Biotin to form the biotinoyl-5'adenosine (Biotinoyl-5'-AMP) in the presence of ATP and specifically link biotin to the lysine residue of the Avi tag (GLNDIFEAQKIEWHE), resulting in fast and efficient biotin labeling of the target protein. However, due to BirA* undergoes the R118G mutation, its activated biotinyl-5'adenylate no longer specifically modifies the Avi-tagged lysine residue, but allows biotin labeling of any exposed protein lysine residue adjacent to it (~10 nm).
Figure 1. The working principle of Beyotime's BioID.
Based on the origin of the biotin ligase, BioID can be further divided into BioID (Escherichia coli), BioID2 (Aquifex aeolicus), AirID (engineered Escherichia coli by an ancestral enzyme reconstruction algorithm with metagenomics), TurboID and mini TurboID (engineered Escherichia coli by yeast surface display), and BASU (Bacillus subtilis).
Table 1. Comparison of some types of BioID.
| Source | Optimal Reaction Temperature | Characteristics | |
| BioID | BirA mutant fromE.coli | 37℃ | Wide applicationw; high sensitivity; suitable for proteins that are poorly soluble or difficult to purify; labeled proteins are easy to purify and have low background. |
| BioID2 | BirA mutant from Aquifex aeolicus | 37-55℃ | Better biotin labeling efficacy; lower biotin concentration; broader reaction temperature range. |
| AirID | Artificially modified E.coli BirA | 26-37℃ | Less cytotoxicity; lower biotin concentration; higher enzyme activity; broader reaction temperature. |
| miniTurboID | Truncated and mutated version of TurboID | 30℃（16~37℃） | Better biotin labeling efficacy; higher biotin ligase activity; broader reaction temperature range. |
To better assist scientific research, Beyotime has constructed N-terminal or C-terminal tagged BioID, AirID, and miniTurboID plasmids and their corresponding control plasmids.
Product Information:
| Cat. No. | Product Name | Pack Size | Price |
| D3021-1µg/100µg | pCMV-N-Flag-BioID2 | 1µg/100µg | / |
| D3023-1µg/100µg | pCMV-C-BioID2-Flag | 1µg/100µg | / |
| D3025-1µg/100µg | pCMV-BioID2-Flag | 1µg/100µg | / |
| D3027-1µg/100µg | pCMV-N-Flag-AirID | 1µg/100µg | / |
| D3029-1µg/100µg | pCMV-C-AirID-Flag | 1µg/100µg | / |
| D3030-1µg/100µg | pCMV-AirID-Flag | 1µg/100µg | / |
| D3034-1µg/100µg | pCMV-N-Flag-miniTurboID | 1µg/100µg | / |
| D3035-1µg/100µg | pCMV-C-miniTurboID-Flag | 1µg/100µg | / |
| D3037-1µg/100µg | pCMV-miniTurboID-Flag | 1µg/100µg | / |
| D3039-1µg/100µg | pCMV-λN-NES-miniTurbo-Flag | 1µg/100µg | / |
APEX (Engineered Ascorbate Peroxidase)
APEX (Ascorbate Peroxidase) is engineered based on ascorbate enzymes derived from pea or soybean. In the presence of H2O2, APEX rapidly converts biotin-tyramide (Biotin-phenol), Biotin-4-aminophenol or Biotin-Naphthylamine (Biotin- Nap) into radicals that can label any exposed electron-rich amino acid residues (Tyr, Trp, Cys or His) within a 20 nm radius of either directly interacting proteins or neighboring proteins. The biotin-labeled proteins can be purified from cell lysates by Streptavidin magnetic beads or gels and identified by mass spectrometry. In addition, APEX is also often used for electron microscopy (EM) cell imaging, hence also named as EM tag. The principle is that fixed cells expressing APEX fusion proteins catalyze the oxidative polymerization of DAB to generate a local precipitate in the presence of H2O2. The polymerized DAB is subsequently stained with dense electron-rich OsO4 to produce the EM contrast that can be imaged by electron microscopy.
APEX2 is an improved and enhanced version of APEX. The advantages of APEX2 over APEX are: (i) Higher expression of APEX2 fusion proteins with better stability in mammalian cells; (ii) Higher enzyme activity of APEX2, thus allowing more efficient neighboring protein biotin labeling and electron microscopy imaging of cells even with lower expression of APEX2.
Based on APEX2, Beyotime has produced pCMV-N-NES-Flag-APEX2 and pCMV-N-mito-Flag-APEX2 plasmids.
pCMV-N-NES-Flag-APEX2 is a plasmid for the expression of APEX2 proteins fused with nuclear export signal (NES) and Flag tag at the N terminus in mammalian cells for biotin labeling of target proteins interacting with APEX2. In the presence of H2O2, APEX2 rapidly converts biotin-based tyramide, biotin-4-aminophenol or biotin-naphthylamine into radicals within 1 min. These radicals can label any exposed electron-rich amino acid residues (Tyr, Trp, Cys or His) within a 20 nm radius of either directly interacting proteins or neighboring proteins which can subsequently be isolated and purified from cell lysates by Streptavidin magnetic beads or gels and identified by mass spectrometry (Figure 2).
pCMV-N-mito-Flag-APEX2 is a plasmid for the expression of APEX2 proteins fused with mitochondrial matrix target peptide (mito) and Flag tag at the N terminus in mammalian cells for biotin labeling of target proteins interacting with APEX2. Please refer to pCMV-N-NES-Flag-APEX2 for the working principle of this plasmid. It can play a crucial role in screening and identifying protein-protein interactions within mitochondria and exploring related functions.
Figure 2. The working principle of Beyotime’s pCMV-N-NES-Flag-APEX2
Product Information:
| Cat. No. | Product Name | Pack Size | Price |
| D3044-1µg /100µg | pCMV-N-NES-Flag-APEX2 | 1µg/100µg | / |
| D3047-1µg/100µg | pCMV-N-mito-Flag-APEX2 | 1µg/100µg | / |
RaPID (RNA-Protein Interaction Detection)
RaPID (RNA-Protein Interaction Detection) is an in vivo RNA-centric technique that uses biotin ligases to biotin-label proteins interacting with RNA decoy fragments, followed by isolation and purification of biotin-labeled proteins from cell lysates by Streptavidin magnetic beads or gels, and identification of RNA-interacted proteins by mass spectrometry or Western blot (Figure 3). RaPID plays a critical role in screening and characterizing the interactions of noncoding RNA sequences (including Long noncoding RNA (lncRNA), Small nucleolar RNA (snoRNA) and Untranslated mRNA region) with RNA-binding proteins (RBP) in live cells and exploring related functions.
RaPID consists of 2 plasmids: the RaPID biotin ligase vector (pCMV-λN-NES-miniTurbo-Flag) for expressing RaPID protein and the RNA decoy vector (pCMV-EGFP-BoxB-RNAmotif-BoxB) for expressing RNA component.
pCMV-λN-NES-miniTurbo-Flag is a plasmid for fusion expression of λN peptide fragment, nuclear exocytosis signal and miniTurbo in mammalian cells for biotin-labeling of target proteins that interact with RNA decoy fragments.
pCMV-EGFP-BoxB-RNAmotif-BoxB is a vector for expressing RNA decoy fragments in mammalian cells. The RNA decoy fragments are sandwiched by 2 BoxB RNA stem-loop structures of λ phage, where the BoxB stem-loop structure has a high affinity for the λN peptide segment.
Figure 3. The working principle of Beyotime’s RaPID.
Product Information:
| Cat. No. | Product Name | Pack Size | Price |
| D3039-1µg /100µg | pCMV-λN-NES-miniTurbo-Flag | 1µg/100µg | 888.00元/1169.00元 |
| D3040-1µg/100µg | pCMV-EGFP-BoxB-RNAmotif-BoxB | 1µg/100µg | / |
| D3042-1µg/100µg | pCMV-EGFP-BoxB-EDEN15-BoxB | 1µg/100µg | / |