Strep Tag II Monoclonal Antibody

What is Strep Tag II and how does it function in experimental systems?

Strep Tag II is a synthetic peptide tag (sequence: NWSHPQFEK) that can be strategically incorporated into recombinant proteins for detection, purification, and functional studies. It functions as a receptor-intrinsic surface marker that enables multiple downstream applications including identification, rapid purification, and selective expansion of tagged cells or proteins. When incorporated into chimeric antigen receptors (CARs) or T cell receptors (TCRs), Strep Tag II provides engineered T cells with a reliable marker that maintains receptor functionality while adding experimental versatility .

The tag's relatively small size (8 amino acids) minimizes interference with protein folding and function while providing sufficient accessibility for antibody recognition. For optimal functionality, Strep Tag II is typically flanked by glycine/serine-rich linker sequences that enhance flexibility and surface accessibility without compromising the tagged protein's native structure .

What are the primary applications of Strep Tag II monoclonal antibodies in protein research?

Strep Tag II monoclonal antibodies serve multiple critical functions in research settings:

• Protein Detection: They enable specific detection of Strep-tagged proteins in various assay formats including Western blotting, immunoprecipitation, and immunofluorescence .

• Cell Tracking: Anti-Strep Tag II antibodies can be used to identify and track cells expressing Strep-tagged receptors both in vitro and in vivo, allowing researchers to monitor cellular distribution and persistence over time .

• Purification Processes: These antibodies facilitate the isolation of Strep-tagged cells or proteins from heterogeneous populations, enabling the generation of highly purified samples for downstream applications .

• Selective Activation: When coupled to microbeads along with co-stimulatory antibodies (e.g., anti-CD28), anti-Strep Tag II antibodies can selectively activate and expand cells expressing Strep-tagged receptors, providing a method for generating large numbers of engineered cells while maintaining their functional properties .

How should researchers optimize immunodetection protocols for Strep Tag II?

Optimizing immunodetection protocols for Strep Tag II requires careful consideration of several experimental parameters:

• Antibody Selection: Choose monoclonal antibodies specifically validated for the intended application (Western blot, immunoprecipitation, flow cytometry, etc.). Clones like GT661 have been extensively validated across multiple applications .

• Antibody Concentration: Determine optimal antibody concentration through titration experiments. For immunoprecipitation, approximately 2.5 μg of anti-Strep Tag II antibody per sample has proven effective for capturing Strep-tagged proteins .

• Tag Accessibility: Consider the structural context of the Strep Tag II within the target protein. Tags positioned at termini or within flexible regions generally show better antibody accessibility compared to those in structurally constrained regions .

• Detection Method: For flow cytometry applications, fluorophore-conjugated anti-Strep Tag II antibodies provide direct detection, while biotinylated antibodies followed by fluorophore-conjugated streptavidin can amplify signal for weakly expressed targets .

• Controls: Always include appropriate controls including untagged versions of your protein of interest and irrelevant Strep-tagged proteins to confirm specificity of detection .

How can Strep Tag II be strategically positioned for optimal functionality in receptor engineering?

Strategic positioning of Strep Tag II within engineered receptors significantly impacts both tag detection and receptor function. Research has demonstrated several successful approaches:

• NH₂-Terminal Placement: Positioning Strep Tag II at the amino terminus of receptors generally provides good accessibility for antibody binding while minimally affecting receptor function .

• Inter-Domain Insertion: Incorporation between variable light (VL) and variable heavy (VH) domains can provide excellent tag exposure while maintaining proper domain folding. This approach requires careful design of flanking glycine/serine-rich linkers to prevent structural constraints .

• Spacer Region Integration: Introducing Strep Tag II between the single-chain variable fragment (scFv) and hinge/spacer regions of CARs has proven particularly effective. This positioning not only facilitates antibody access but in some cases enhances receptor signaling, potentially due to added flexibility in the spacer region .

• Multiple Tag Integration: For applications requiring enhanced detection sensitivity or stronger binding to purification matrices, incorporating multiple Strep Tag II sequences (up to three) has proven beneficial. Flow cytometry data demonstrates that staining intensity increases with the number of Strep Tag II sequences incorporated, with triple-tagged receptors showing the highest detection levels .

Importantly, functional studies demonstrate that properly designed Strep-tagged receptors maintain equivalent or sometimes enhanced cytolytic activity and cytokine production compared to their untagged counterparts .

What methodological approaches enable selective expansion of Strep Tag II-labeled engineered T cells?

Selective expansion of Strep Tag II-labeled engineered T cells can be achieved through specialized methodological approaches:

• Antibody-Coated Microbeads: Magnetic microbeads coated with anti-Strep Tag II monoclonal antibodies can selectively bind and activate cells expressing Strep-tagged receptors. Optimal protocols use approximately 150 μg of anti-Strep Tag II antibody per 50 mg of beads .

• Co-stimulatory Signal Integration: Combining anti-Strep Tag II with co-stimulatory antibodies (particularly anti-CD28) on microbeads provides both primary activation and co-stimulatory signals required for robust T cell proliferation. A ratio of 150 μg anti-Strep Tag II antibody to 50 μg anti-CD28 antibody has demonstrated significant efficacy .

• Expansion Kinetics: This approach typically yields >100-fold expansion of Strep Tag II-expressing T cells within 9 days of culture, with sequential stimulations enabling up to 10⁶-fold expansion while maintaining a diverse T cell receptor repertoire .

• Enrichment Effect: During expansion, the proportion of Strep Tag II-expressing cells increases substantially (from approximately 26-33% to 84-92%), creating a highly enriched population without requiring additional purification steps .

• Phenotype Preservation: T cells expanded through this method maintain critical phenotypic markers including CD62L, CD27, and CD28, which are associated with enhanced persistence and therapeutic efficacy .

This methodology provides significant advantages over conventional expansion approaches that utilize anti-CD3/CD28 beads, which activate and expand both transduced and non-transduced T cells indiscriminately .

How can Strep Tag II monoclonal antibodies facilitate reisolation and functional analysis of engineered cells in vivo?

Anti-Strep Tag II monoclonal antibodies enable powerful approaches for tracking, reisolation, and functional analysis of engineered cells in vivo:

• In Vivo Tracking: Fluorophore-conjugated anti-Strep Tag II antibodies permit identification of CAR-T or TCR-T cells in blood and tissue samples collected during in vivo studies. This approach allows monitoring of cellular persistence, expansion, and contraction dynamics throughout therapeutic responses .

• High-Purity Cell Isolation: Following in vivo administration, engineered cells can be reisolated from blood or tissue samples using anti-Strep Tag II antibodies with >95% purity. This high-purity isolation enables downstream molecular analyses without significant contamination from non-engineered cells .

• Molecular Profiling: Reisolated cells can undergo comprehensive molecular characterization including gene expression analysis, cytokine profiling, and functional assessments to understand their in vivo activation state and potential exhaustion markers .

• Comparative Analysis: Side-by-side comparison of engineered cells before infusion and after reisolation provides critical insights into phenotypic and functional changes occurring in the in vivo environment. Research has demonstrated significant upregulation of IFN-γ, IL-2, TNF-α, GM-CSF, IL-13, and IL-5 in CAR-T cells isolated from tumor-bearing versus non-tumor-bearing hosts .

• Technical Compatibility: Anti-Strep Tag II antibodies have demonstrated reliable performance in detection of engineered cells in complex biological matrices including peripheral blood mononuclear cell (PBMC) preparations and whole blood samples, facilitating clinical translation of these approaches .

These methodological capabilities are particularly valuable for investigating mechanisms of therapeutic efficacy or failure in adoptive cell therapy studies .

What are the potential immunogenicity considerations when using Strep Tag II in therapeutic applications?

The potential immunogenicity of Strep Tag II represents an important consideration for therapeutic applications, particularly for engineered cell therapies:

• Epitope Analysis: Computational prediction using algorithms like NetMHC3.4 can identify potential human leukocyte antigen (HLA)-binding peptides within the Strep Tag II sequence and flanking regions. Initial screening has not identified sequences likely to bind common HLA class I alleles with high affinity, suggesting limited T cell immunogenicity .

• Multiple Foreign Elements: Engineered receptors containing Strep Tag II typically include additional foreign elements such as murine single-chain variable fragments (scFvs) and artificial fusion sites. The cumulative immunogenic potential of these elements requires careful assessment .

• Humoral Immunity: While T cell epitope prediction provides insights into cellular immunity, potential antibody responses against Strep Tag II can only be fully evaluated through in vivo studies in immunocompetent hosts .

• Design Optimization: Strategic positioning of the tag in less exposed regions or incorporation of flanking sequences that minimize generation of novel epitopes may reduce immunogenic potential .

• Monitoring Strategy: For clinical applications, immunomonitoring protocols should include assessment of both cellular and humoral responses against the Strep Tag II component to identify potential immunogenicity issues early in treatment .

Importantly, preclinical evaluation in immunodeficient mouse models cannot fully address these immunogenicity concerns, highlighting the need for careful translational studies when moving toward clinical applications .

How does the number and positioning of Strep Tag II sequences affect detection sensitivity and purification efficiency?

The number and positioning of Strep Tag II sequences significantly impact both detection sensitivity and purification efficiency of tagged proteins or cells:

• Detection Sensitivity Correlation: Flow cytometry data demonstrates a direct correlation between the number of Strep Tag II sequences and detection sensitivity. Cells expressing CARs with three Strep Tag II sequences show substantially higher staining intensity with anti-Strep Tag II antibodies compared to those with single tags .

• Position-Dependent Accessibility: The structural context surrounding the Strep Tag II significantly affects antibody access. Tags positioned in flexible regions (such as between domains or at termini) generally show superior detection compared to those in structurally constrained positions .

• Purification Threshold Effect: For purification applications, a minimum binding strength threshold must be achieved. Research demonstrates that while CARs containing three Strep Tag II sequences can be efficiently purified using StrepTactin beads, those with single tags show inadequate enrichment, suggesting that the affinity of a single Strep Tag II is insufficient for stable binding during purification workflows .

• Spacer Length Considerations: The addition of Strep Tag II sequences effectively increases the spacer length in CAR constructs (by approximately 19 amino acids for a single tag). This structural modification can impact receptor signaling independent of the tag's functional properties, potentially enhancing cytokine production and proliferative responses .

• Functional Impact Assessment: When optimizing tag number and position, comprehensive functional assessment is essential. Properly designed multi-tagged receptors maintain or enhance cytolytic function, cytokine production, and proliferative capacity compared to conventional untagged receptors .

These findings highlight the importance of rational design when incorporating Strep Tag II into proteins for experimental or therapeutic applications.

What purification techniques maximize recovery of Strep Tag II-labeled proteins and cells?

Optimizing purification of Strep Tag II-labeled proteins and cells requires consideration of several methodological approaches:

• T-CATCH Rapid Isolation: The T cell Affinity Column for Capture of Tagged Hybrid-receptors (T-CATCH) system enables high-efficiency isolation of cells expressing multi-Strep-tagged receptors. This approach allows processing of large cell numbers with minimal manipulation time, preserving cellular viability and functionality .

• StrepTactin Matrix Selection: For protein purification, engineered StrepTactin matrices provide higher affinity for Strep Tag II compared to streptavidin. The specific matrix selection should be optimized based on the number of Strep tags incorporated and the complexity of the starting material .

• Elution Condition Optimization: When purifying proteins, desthiobiotin-based elution provides gentle, competitive displacement of Strep-tagged proteins while preserving their structural integrity and functional properties. For cell isolation, release from capture matrices must be optimized to maintain cellular viability .

• Multi-Tag Incorporation: As demonstrated with CAR-T cells, proteins with three Strep Tag II sequences show significantly higher purification efficiency compared to those with single tags. This approach is particularly valuable when high purity is required .

• Functional Preservation Assessment: Following purification, thorough functional assessment is critical. Properly isolated Strep Tag II-labeled CAR-T cells demonstrate equivalent tumor elimination capacity compared to conventionally prepared cells, whereas cells isolated through alternative methods (such as anti-EGFR antibody sorting) show compromised in vivo persistence and efficacy without additional recovery culture .

These methodological considerations highlight the importance of technique selection and optimization based on the specific experimental or therapeutic requirements.

How does Strep Tag II compare with alternative tagging systems for cell therapy applications?

When evaluating tagging systems for cell therapy applications, Strep Tag II offers distinct advantages and limitations compared to alternative approaches:

This comparative analysis highlights the particular value of Strep Tag II in applications requiring multifunctional capabilities and minimal structural perturbation.

What are emerging research directions for expanding Strep Tag II utility in cellular therapeutics?

Several innovative research directions are expanding the utility of Strep Tag II in cellular therapeutics:

• Universal Manufacturing Platforms: Integration of Strep Tag II into universal receptor designs could streamline manufacturing workflows by enabling standardized purification, activation, and expansion protocols regardless of the specific antigen target .

• Regulatable Cell Therapies: Incorporation of Strep Tag II into regulatable receptor systems could enable selective activation or depletion of therapeutic cells through administration of anti-Strep Tag II antibodies or antibody-drug conjugates, providing an additional layer of safety control .

• Combinatorial Antigen Recognition: Engineering of multi-component receptor systems where Strep Tag II-mediated interactions contribute to the assembly of functional signaling complexes could enhance specificity and reduce off-target effects of cellular therapeutics .

• Non-Invasive Imaging Applications: Development of radiolabeled or otherwise modified anti-Strep Tag II antibodies could enable non-invasive tracking of cellular therapeutics through imaging modalities such as PET or SPECT, facilitating monitoring of cell distribution and persistence .

• Immunologically Stealth Variants: Computational design and experimental validation of Strep Tag II variants with reduced immunogenicity could address potential concerns regarding immune recognition while maintaining functional properties .

These emerging directions highlight the continued evolution of Strep Tag II applications beyond current capabilities, potentially addressing key challenges in the development and clinical implementation of engineered cellular therapeutics .