This article details the molecular characteristics of recombinant STAT6 protein with dual tags (His & Strep), its expression and purification system, key applications in JAK-STAT signaling pathway research, and standardized experimental protocols based on its tags, providing an efficient tool for signal transduction and immunology studies.

STAT6 (Signal Transducer and Activator of Transcription 6) is a key member of the JAK-STAT signaling pathway, playing a central role in IL-4- and IL-13-mediated immune responses, regulating processes such as B cell proliferation, Th2 cell differentiation, and allergic reactions. For in vitro studies, recombinant STAT6 His&Strep Tag protein has become an essential tool for investigating its DNA binding, phosphorylation regulation, and protein interaction mechanisms.

I. Molecular Construction and Expression System Characteristics

Typical commercial or self-constructed recombinant STAT6 proteins employ a dual-affinity tag strategy to optimize purification and detection efficiency.

Core Domains:

Usually include the full-length STAT6 or functional truncations (e.g., containing DNA-binding domain, SH2 domain, and transcriptional activation domain).

Tag Configuration:

Tandem His-tag (6xHis) and Strep-tag II are fused to the N- or C-terminus of the protein.

His-tag:

Provides efficient and robust binding to nickel (Ni²⁺) or cobalt (Co²⁺) chelate resins, suitable for initial capture and purification under denaturing or non-denaturing conditions.

Strep-tag II:

Binds with high affinity and specificity to Strep-Tactin resin, which can be competitively eluted under mild conditions using biotin. This step often serves as a subsequent purification step, yielding highly pure protein while maintaining STAT6's native conformation and activity.

Expression System:

Primarily expressed in insect cell-baculovirus systems (e.g., Sf9 cells) or mammalian cells (e.g., HEK293). These systems possess eukaryotic post-translational modification capabilities, ensuring STAT6 undergoes critical modifications such as phosphorylation (albeit at low basal levels, providing an ideal substrate for activation studies) and dimerization, making its function closer to the native state.

II. Core Applications in Signal Transduction Research

Dual-tagged recombinant STAT6 protein provides highly controllable experimental material for dissecting JAK-STAT pathway mechanisms.

In Vitro Phosphorylation and Kinase Assays:

Purified STAT6 protein can serve as a substrate, co-incubated with purified JAK kinases (e.g., JAK1, JAK3) or cell lysates containing activated receptors, to directly study phosphorylation kinetics, site specificity, and inhibitor effects.

DNA-Binding Activity Analysis (EMSA):

Dual-tag purified STAT6, activated (phosphorylated) in vitro, can be used in gel shift or electrophoretic mobility shift assays to quantitatively analyze its binding ability and specificity to specific DNA response elements (e.g., GAS or IL-4 response elements in target gene promoters).

Protein-Protein Interaction Studies:

Pull-down / Affinity Purification:

Using His or Strep tags, recombinant STAT6 is immobilized on resin to "fish" interacting proteins (e.g., co-activators, other transcription factors, phosphatases) from cell lysates or protein libraries.

Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

STAT6 is fixed on biosensor chips via tags to analyze binding kinetics (Kon, Koff, KD values) with potential ligands (e.g., phosphorylated receptor intracellular segments, chaperone proteins) in real-time and quantitatively.

Structural Biology Research:

Highly pure and homogeneous dual-tagged STAT6 protein serves as foundational material for X-ray crystallography or cryo-EM analysis to resolve its three-dimensional structure with DNA or interacting protein complexes.

III. Advantages of Standardized Experimental Protocols Based on Dual Tags

Tandem purification yields high-purity protein: Sequential immobilized metal affinity chromatography (IMAC) and Strep-Tactin affinity chromatography nearly completely remove host cell proteins, nucleic acids, and other impurities, meeting high-sensitivity experimental requirements.

High Flexibility:

Researchers can choose elution methods based on downstream applications. For maintaining native protein state, biotin elution of Strep-tag is preferred; for analysis under denaturing conditions, imidazole (His-tag) or low-pH buffers can be used.

Convenient Detection and Immobilization:

Both tags have commercially available high-sensitivity, high-specificity detection antibodies (anti-His, anti-Strep), facilitating Western Blot quantification and validation. The tags also provide a universal interface for directional immobilization on various carriers (chips, microspheres, ELISA plates).

IV. Key Research Examples and Technical Points

Investigating STAT6 Activation Mechanisms:

Parallel in vitro kinase assays and EMSA with purified wild-type and mutant (e.g., DNA-binding domain or SH2 domain mutants) STAT6 His&Strep proteins can precisely identify functionally critical amino acid residues.

Screening Small-Molecule Inhibitors:

Establishing an in vitro phosphorylation detection system with purified STAT6 protein and JAK kinases is an effective platform for screening compounds that block this specific node in the signaling pathway.

Technical Points:

Storage and Activity:

Recommended storage in non-denaturing buffers containing reducing agents (e.g., DTT) and glycerol at -80°C in aliquots to avoid repeated freeze-thaw cycles.

Tag Removal Option: Some vectors include protease cleavage sites (e.g., TEV, HRV 3C) between the tags and STAT6, allowing tag removal post-purification for tag-sensitive studies.

Conclusion

Recombinant STAT6 His&Strep Tag protein, through its ingenious dual-tag design, achieves high-yield, high-purity preparation and versatile application potential. It greatly facilitates in vitro biochemical and structural studies of STAT6 in transcriptional regulation, cell signal transduction, and particularly the molecular mechanisms of Th2-type immune responses and related diseases (e.g., asthma, allergies), serving as a standardized key reagent for researchers in immunology, cell biology, and drug development.

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