Strep-Tag Monoclonal Antibody

What is Strep-tag II and how does it function in protein expression systems?

Strep-tag II is a synthetic octapeptide with the amino acid sequence WSHPQFEK that serves as a widely used tag in protein expression applications. This tag enables specific recognition by anti-Strep-tag monoclonal antibodies, facilitating detection, purification, and immobilization of recombinant fusion proteins . The Strep-tag system functions through highly specific molecular recognition, with different antibody clones exhibiting varying binding characteristics. For example, StrepMAB-Immo demonstrates near-irreversible binding of Strep-tag II when N-terminally extended by SerAla, making it particularly valuable for applications requiring stable interactions .

In experimental workflows, Strep-tag II can be positioned at either the N- or C-terminus of a target protein, offering flexibility in experimental design while minimizing interference with protein structure and function. This versatility makes it an ideal research tool for multiple protein analysis applications beyond simple detection.

What distinguishes the different anti-Strep-tag monoclonal antibody clones?

Different anti-Strep-tag monoclonal antibody clones have been developed for specific research applications:

StrepMAB-Immo (clone Strep-MAB) demonstrates particularly strong binding affinity, making it ideal for applications where stable interactions are required, such as immobilization of target proteins in ELISA and biosensor applications . In contrast, StrepMAB-Classic (clone Strep-tag II) recognizes the Strep-tag II sequence in both N-terminal and C-terminal positions, making it particularly suitable for Western blot applications where antibody accessibility to the tag in different conformational contexts is important .

How does the Strep-tag system compare to other affinity tagging systems?

The Strep-tag system offers several methodological advantages in protein research compared to alternative tagging systems. Its relatively small size (8 amino acids) minimizes interference with protein folding and biological activity. The system provides a centralized platform for multiple research applications including protein purification, detection, and functional analysis .

When implementing the Strep-tag system, researchers should consider that the optimal positioning of the tag may vary depending on the specific protein structure and the intended application. Experimental validation is recommended to ensure tag accessibility while maintaining protein function.

How can Strep-tag II be strategically positioned in receptor design for optimal detection and functionality?

The strategic positioning of Strep-tag II in receptor design requires consideration of both molecular structure and functional requirements. Research has demonstrated successful incorporation of Strep-tag II at multiple positions within chimeric antigen receptors (CARs), including:

• At the NH₂ terminus

• Between the variable light (VL) and variable heavy (VH) regions

• Between the single-chain variable fragment (scFv) and the hinge region

Experimental data indicate that all these positioning strategies maintain receptor functionality while enabling detection with anti-Strep-tag II monoclonal antibodies. Notably, incorporation of multiple Strep-tag II sequences (up to three) significantly enhances detection sensitivity without compromising receptor function .

When designing Strep-tagged receptors, researchers should incorporate glycine/serine linkers flanking the Strep-tag sequence to minimize steric hindrance and ensure proper folding. This approach has been validated with various receptor types including CD19-specific CARs with different costimulatory domains (4-1BB/CD3ζ or CD28/CD3ζ) .

How does Strep-tag II contribute to adoptive T cell therapy development?

Strep-tag II incorporation provides several critical advantages in adoptive T cell therapy development:

• Identification and tracking: Anti-Strep-tag II antibodies enable precise identification of engineered T cells in both in vitro and in vivo settings. Studies have demonstrated that Strep-tag CAR-T cells can be readily detected in blood samples after infusion, allowing monitoring of their proliferation and contraction during tumor elimination .

• Purification capability: The tag facilitates rapid purification of engineered T cells to >95% purity using anti-Strep-tag II monoclonal antibodies, enabling detailed analysis of gene expression changes during antitumor responses .

• Selective expansion: Microbeads coated with anti-Strep-tag II antibodies (alone or combined with anti-CD28) induce selective activation and expansion of Strep-tag CAR-T cells, increasing their frequency from ~26-33% to 84-92% in culture systems .

• Maintained functionality: Importantly, Strep-tag incorporation does not compromise the antitumor activity of engineered T cells. Studies in NSG mice engrafted with Raji lymphoma have shown that T cells expressing CD19 CARs containing one or three Strep-tag II sequences eliminated tumors as effectively as conventional CAR-T cells .

What molecular mechanisms underlie Strep-tag II detection and binding?

The molecular interaction between Strep-tag II and anti-Strep-tag monoclonal antibodies involves specific recognition of the WSHPQFEK sequence. For StrepMAB-Immo, the binding is enhanced when the tag is N-terminally extended by SerAla, leading to near-irreversible interactions that are particularly valuable for stable immobilization applications .

The binding kinetics and affinity of different anti-Strep-tag antibody clones vary substantially, influencing their suitability for specific applications. Researchers should select the appropriate antibody clone based on experimental requirements:

• Use StrepMAB-Immo for applications requiring stable, near-irreversible binding

• Use StrepMAB-Classic for applications requiring detection of both N- and C-terminal tags, especially in Western blot applications

What are the optimal protocols for reconstituting and storing Strep-tag antibodies?

Proper reconstitution and storage of Strep-tag antibodies is critical for maintaining their activity and specificity. Follow these methodological guidelines:

For StrepMAB-Immo (clone Strep-MAB):

• Reconstitute the lyophilized antibody with 0.1 ml PBS

• Take care during reconstitution as the protein may appear as a film at the bottom of the vial

• Gently mix after reconstitution to ensure complete dissolution

• For long-term storage, add 0.09% sodium azide

• After reconstitution, the antibody concentration will be approximately 1.0 mg/ml

For StrepMAB-Classic (clone Strep-tag II):

• Reconstitute the lyophilized antibody with 0.2 ml water

• For long-term storage, add 0.09% sodium azide

• After reconstitution, the antibody concentration will be approximately 0.5 mg/ml

Both antibodies are purified IgG1 isotype antibodies. StrepMAB-Immo is prepared by ammonium sulfate precipitation from tissue culture supernatant, while StrepMAB-Classic is purified by affinity chromatography on Protein G .

How can Strep-tag technology be utilized for monitoring engineered T cell expansion and function?

Strep-tag technology provides powerful methodological approaches for monitoring engineered T cell expansion and function:

• In vitro monitoring:

  • Anti-Strep-tag II antibody staining can detect CAR expression on T cell surfaces regardless of the position or number of Strep-tag II sequences

  • Multiple Strep-tag II sequences (up to three) enhance detection sensitivity

• In vivo tracking:

  • Strep-tag CAR-T cells can be tracked in blood samples after infusion using anti-Strep-tag II antibody staining

  • The approach allows monitoring of proliferation and contraction kinetics during tumor elimination

• Functional analysis:

  • Strep-tag CAR-T cells can be isolated from blood after transfer using anti-Strep-tag II antibodies

  • This enables detailed analysis of gene expression changes during antitumor responses

  • Studies have demonstrated upregulation of IFN-γ, IL-2, TNF-α, GM-CSF, IL-13, and IL-5 in Strep-tag CAR-T cells from tumor-bearing mice compared to non-tumor bearing controls

• Detection in clinical samples:

  • Anti-Strep-tag II antibodies can readily detect CAR-T cells spiked into peripheral blood mononuclear cells (PBMCs) and whole blood

  • This capability facilitates potential clinical monitoring applications

What protocols enable selective expansion of Strep-tag engineered T cells?

Selective expansion of Strep-tag engineered T cells can be achieved using the following protocol:

• Prepare microbeads coated with anti-Strep-tag II antibody alone or combined with anti-CD28 antibody

• Co-culture Strep-tag CAR-T cells with the antibody-coated microbeads

• Monitor activation by assessing CD25 upregulation (which occurs only in Strep-tag CAR-T cells)

• Continue culture for approximately 9 days to achieve >100-fold expansion of both CD4+ and CD8+ Strep-tag CAR-T cells

• This approach increases the frequency of Strep-tag CAR-T cells from ~26-33% to 84-92%

Important methodological considerations:

• This expansion protocol works independently of scFv specificity and costimulatory domains in the CAR

• T cells expanded with this method maintain a diverse TCR repertoire

• Expanded cells continue to express important markers including CD62L, CD28, and CD27

• The cells maintain CAR-directed cytolytic function and proliferate in response to tumor cells

• For applications requiring large cell numbers, repeated stimulation with anti-Strep-tag II/CD28 beads every 9-12 days can achieve up to 1×10^6-fold expansion

What factors affect Strep-tag detection sensitivity and how can they be optimized?

Several factors can influence Strep-tag detection sensitivity:

• Tag accessibility: The position of the Strep-tag II within the protein structure significantly affects its accessibility to antibodies. Consider testing multiple positioning strategies when designing Strep-tagged constructs.

• Number of tag repeats: Research has demonstrated that incorporating multiple Strep-tag II sequences (up to three) enhances detection sensitivity. The staining intensity is highest for constructs containing three Strep-tag II sequences .

• Antibody clone selection: Different anti-Strep-tag antibody clones have varying binding characteristics. StrepMAB-Immo demonstrates near-irreversible binding when the tag is N-terminally extended by SerAla, while StrepMAB-Classic recognizes both C- and N-terminal tags .

• Buffer conditions: Ensure optimal buffer conditions during antibody-antigen interactions. The absence of specific buffer solutions or preservative stabilizers in reconstituted antibodies may affect their performance .

• Sample preparation: Proper sample preparation is critical, especially for applications like Western blotting. Note that StrepMAB-Immo is not recommended for Western blot detection of Strep II fusion proteins .

How can researchers validate the functionality of Strep-tagged proteins?

Validating the functionality of Strep-tagged proteins requires comprehensive testing to ensure that tag incorporation does not interfere with biological activity:

• Comparative functional assays: Compare the activity of Strep-tagged constructs with untagged versions to assess potential functional impacts. For example, studies with Strep-tag CAR-T cells demonstrated equivalent cytolytic activity against target tumor cells compared to conventional CARs .

• Specificity testing: Confirm that Strep-tagged constructs maintain proper target specificity. CAR-T cells with Strep-tag modifications should lyse target-positive cells (e.g., K562/CD19 and CD19+ Raji cells) but not control cells (e.g., K562/ROR1) .

• Cytokine production analysis: Assess whether Strep-tagged constructs maintain appropriate signaling capability by measuring cytokine production after target engagement. Strep-tag CAR-T cells should produce cytokines like IL-2 and IFN-γ after co-culture with target-positive tumor cells .

• In vivo functional validation: When possible, validate functionality in relevant in vivo models. Studies have confirmed that Strep-tag CAR-T cells eliminate tumors in mouse models with similar efficacy to conventional CAR-T cells .

How is Strep-tag technology advancing immunotherapy development?

Strep-tag technology is significantly enhancing immunotherapy development through several innovative applications:

• Simplified manufacturing: The incorporation of Strep-tag II into synthetic antigen receptors facilitates identification and rapid purification of engineered T cells, potentially streamlining cGMP manufacturing of adoptive T cell therapies .

• Selective expansion strategies: Anti-Strep-tag II antibody-coated microbeads enable selective expansion of receptor-bearing T cells, addressing the current limitation of mixed products containing both transduced and non-transduced cells .

• Enhanced monitoring capabilities: Strep-tag technology allows for tracking engineered T cells in vivo and their reisolation for detailed functional analysis, providing valuable insights into mechanisms of therapeutic efficacy or failure .

• Broad applicability: The Strep-tag approach has been successfully applied to various receptor types, including CARs with different ligand specificities and costimulatory domains (4-1BB/CD3ζ or CD28/CD3ζ), as well as TCRs .

• Integration with antibody development platforms: The Strep-tag system can centralize various steps in antibody development workflows, making the process more time-efficient and cost-effective .

What are the potential future applications of Strep-tag in protein analysis technologies?

The Strep-tag system has significant potential for advancing protein analysis technologies:

• Integrated protein analysis platforms: The versatility of the Strep-tag makes it suitable as a central component for multiple applications, including protein purification, analysis, immunization, cell sorting, and various assay formats .

• Enhanced biosensor applications: The near-irreversible binding properties of certain Strep-tag antibody interactions make them particularly valuable for developing stable biosensor platforms with improved sensitivity and specificity .

• Advanced antibody development: Strep-tag technology can facilitate various steps in antibody development workflows, including antigen-specific B cell staining and isolation via FACS, ELISA, Western blot, and biopanning applications .

• Therapeutic monitoring innovations: The ability to detect Strep-tagged engineered cells in clinical samples could enable more precise monitoring of cell therapies in patients, potentially improving treatment protocols and outcomes .

These emerging applications underscore the growing importance of Strep-tag technology in advancing protein research and therapeutic development across multiple disciplines.