Myc Tag: Small Tag, Great Significance  

Molecular Characteristics and Structural Basis of the Myc Tag

The Myc tag is a 10 - amino acid peptide (EQKLISEEDL) derived from residues 410 - 419 of the human c - Myc proto - oncoprotein, serving as one of the most widely used epitope tags in molecular biology. This short peptide exhibits unique structural features: negatively charged and polar N - terminal glutamic acid (E410) and glutamine (Q411); a central hydrophobic core (L414 - I415) for structural stability; and a C - terminal aspartic acid (D419) to enhance water solubility. Crystallographic analysis shows that the Myc tag forms a β - turn conformation when binding to its specific antibody (e.g., 9E10), with E412, K413, L414, and E417 constituting key antigenic epitopes. Notably, the Myc tag’s small molecular weight (~1.2 kDa) and neutral isoelectric point (pI≈4.5) minimally affect the structure and function of fusion proteins (retaining >90% activity in most cases), making it an ideal choice for protein labeling. Mass spectrometry studies indicate a half - life of 20 - 24 hours in mammalian cells, with low recognition by the ubiquitin - proteasome system, ensuring labeled protein stability.

Development and Characteristics of Myc Tag Antibodies

The monoclonal antibody 9E10 (IgG1 subtype) against the Myc tag is the earliest and most widely used specific tool antibody. Prepared by hybridoma technology, its antigen - binding site (CDR region) interacts with the Myc tag with high affinity (Kd≈2.4 nM). Epitope mapping reveals that the 9E10 antibody primarily recognizes the EQKLISE sequence of the Myc tag, with mutations in E412 and L414 completely abolishing binding activity. Newer antibodies developed via phage display (e.g., Myc - Tag[4A6]) exhibit higher thermal stability (retaining >95% activity after 30 minutes at 60℃) and a broader pH range (pH 4 - 10). These antibodies show excellent specificity (<0.1% cross - reactivity with endogenous c - Myc) and are suitable for various assays: Western blotting (detection limit 0.1 - 0.5 ng), immunoprecipitation (binding efficiency >90%), immunofluorescence (signal - to - noise ratio >20:1), and flow cytometry (detecting fusion proteins with >1,000 molecules/cell). Engineered nanobodies (e.g., cAbMyc - 1) with smaller sizes (~15 kDa) better recognize Myc - tagged fusion proteins with steric hindrance.

Applications of Myc Tag in Protein Purification Systems

The binding property of the Myc tag to its antibody is widely applied in protein purification. Immunoaffinity purification using 9E10 antibody - conjugated agarose beads yields target proteins with >95% purity under mild elution conditions (e.g., pH 3.0 glycine buffer or competitive Myc peptide), with a recovery rate of 60 - 80%. To enhance purification efficiency, tandem tag systems like Myc/His6 have been developed, combining Ni - NTA resin for rough purification and Myc antibody columns for fine purification, achieving >99% purity. In surface plasmon resonance (SPR), immobilized Myc tags on chips improve data quality due to uniform orientation, reducing non - specific binding by 50 - 70%. Myc - based pull - down assays, combined with mass spectrometry, have become the gold standard for identifying protein complexes, even for low - abundance proteins (<100 copies/cell). Developed Dynabeads Myc - Tag further simplifies operations, completing protein purification from cell lysates within 1 hour, ideal for high - throughput proteomics.

Technical Breakthroughs of Myc Tag in Live Cell Imaging

The Myc tag offers unique advantages in live cell imaging. Fusing the Myc tag with fluorescent proteins (e.g., GFP) and using anti - Myc nanobodies enables dual verification of protein subcellular localization (colocalization rate >90%). Super - resolution microscopy (e.g., STORM) achieves <20 nm localization precision using Myc tag antibody specificity, resolving nanoscale arrangements of postsynaptic density (PSD) proteins in neurons. The developed SunTag system, by tandemly repeating multiple Myc tags (10 - 24 copies), enhances signal intensity by 5 - 10 - fold for single - molecule tracking. Photoactivatable anti - Myc antibodies (e.g., PA - Tag) mark newly synthesized Myc fusion proteins with temporal (±30 seconds) and spatial (≈5 μm activation diameter) precision, aiding studies of protein dynamics (e.g., spatiotemporal regulation of cell cycle - related proteins). Engineered Myc variants (e.g., codon - optimized Myc3×) increase expression by 3 - 5 - fold with <5% cytotoxicity, enabling long - term live cell observation.

Innovative Applications in Gene Therapy and Drug Delivery

Myc tag technology shows broad prospects in gene therapy. In AAV vector design, Myc tags track vector expression (3 - 5 - fold more sensitive than traditional Flag tags) without affecting therapeutic protein activity (retaining >95%). In CAR - T cell therapy, fusing Myc tags with safety switches (e.g., iCasp9) allows >90% modified T cell clearance via antibody - induced dimerization within 30 minutes, controlling cytokine storms. For drug delivery, Myc - labeled exosomes (e.g., Myc - LAMP2B fusions) enable antibody - mediated targeted delivery, enhancing siRNA/small molecule delivery efficiency by 5 - 8 - fold. The developed "protein replacement therapy" fuses functional proteins (e.g., p53) with Myc tags, combined with anti - Myc antibody - transduction domain conjugates (e.g., TAT - MycAb), achieving >80% cellular internalization and 10 - 50 - fold target gene activation. These innovations provide new platforms for precision medicine.

Technical Challenges and Future Directions

Despite wide applications, technical bottlenecks remain: potential interference from endogenous c - Myc (up to 10⁵ molecules/cell in tumor cells) requires more specific antibodies (e.g., those recognizing N - acetylated Myc tags); immunogenicity of mouse - derived 9E10 (half - life 6 - 8 hours in humans) drives humanization (e.g., CDR - grafted HmMyc - 1). Future directions include: developing super - stable Myc variants (e.g., with non - natural amino acids for protease resistance); designing light - controlled reversible anti - Myc tools (e.g., phytochrome - based Myc - Nano); establishing antibody - free detection systems (e.g., Myc - specific aptamers); integrating CRISPR - Cas for site - specific Myc tagging (efficiency >80%). With synthetic biology and protein engineering advances, Myc tag technology will continue providing precise and efficient molecular tools for life science research.

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