Recombinant Mouse C-X-C motif chemokine 11 protein (Cxcl11) (Active)

What is the molecular structure of recombinant mouse CXCL11?

Recombinant mouse CXCL11 is an 8.8 kDa protein (without tags) consisting of 79 amino acid residues. The protein belongs to the intercrine alpha (chemokine CxC) family. When produced recombinantly with an N-terminal His-tag, the molecular weight increases to approximately 12.9 kDa. The amino acid sequence typically begins with FLMFKQGRCLCIGPGMK and continues through to the C-terminal sequence ending with AIEKKNFLRRQ or AIEKKNFLRRQNM depending on the specific construct . The protein contains critical cysteine residues that form disulfide bonds essential for its three-dimensional structure and biological activity. These structural features are preserved in properly folded recombinant versions of the protein produced in expression systems like E. coli .

How does recombinant mouse CXCL11 function in immune regulation?

Recombinant mouse CXCL11, also known as Interferon-gamma-inducible protein 9 (IP-9), functions primarily as a chemotactic factor for activated T cells. It binds with high affinity to the CXCR3 receptor, exhibiting stronger binding than the related chemokines CXCL9 and CXCL10 . This high-affinity binding induces the activation and directed migration (chemotaxis) of T cells toward sites of CXCL11 expression. The protein is produced primarily in response to interferon family cytokines, particularly IFN-γ, placing it within critical inflammatory and immune regulatory pathways . In experimental settings, recombinant CXCL11's activity can be measured by its ability to chemoattract BaF3 cells transfected with human CXCR3, with an effective dose (ED50) typically below 10 ng/mL .

What expression systems are used to produce active recombinant mouse CXCL11?

Recombinant mouse CXCL11 is predominantly produced using Escherichia coli expression systems. This bacterial expression system offers several advantages including high yield, cost-effectiveness, and relatively straightforward purification processes . When expressed in E. coli, the protein is typically tagged with an N-terminal polyhistidine (His) tag to facilitate purification. The resulting recombinant protein exhibits greater than 90% purity as determined by SDS-PAGE analysis . The expression range typically covers amino acids 22-98 of the full sequence . While E. coli is the most common expression system, it's important to note that proper folding and formation of disulfide bonds are critical for functional activity, requiring optimal purification and refolding protocols to ensure the recombinant protein maintains its native structure and biological function .

What are the recommended reconstitution protocols for lyophilized recombinant mouse CXCL11?

Reconstitution of lyophilized recombinant mouse CXCL11 requires careful handling to preserve biological activity. First, briefly centrifuge the vial prior to opening to bring all contents to the bottom. The lyophilized protein should be reconstituted in sterile deionized water to achieve a concentration of 0.1-1.0 mg/mL . It is strongly recommended to add glycerol to a final concentration of 5-50% to stabilize the protein. The optimal final glycerol concentration is typically 50% . After reconstitution, allow the solution to sit at room temperature for at least 20 minutes to ensure complete dissolution. Importantly, vigorous shaking or vortexing should be avoided as this may impair the biological activity of the protein . The reconstituted protein should be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C or -80°C for long-term storage .

How can researchers measure the biological activity of recombinant mouse CXCL11?

The biological activity of recombinant mouse CXCL11 can be assessed through several complementary approaches. The gold standard method involves chemotaxis assays using BaF3 cells transfected with human CXCR3 receptor . In this assay, the ability of CXCL11 to induce directed cell migration is measured, with effective concentrations typically below 10 ng/mL. In addition to chemotaxis assays, receptor binding assays can be performed to determine the affinity of the recombinant protein for CXCR3. For in vivo applications, gene expression analysis using RT-qPCR can confirm the functional expression of CXCL11 . When using CXCL11 in experimental settings, researchers should also measure endotoxin levels, which should be below 0.1 EU per 1 μg of protein as determined by the LAL (Limulus Amebocyte Lysate) method to ensure that observed biological effects are not due to endotoxin contamination .

What storage conditions maximize stability of recombinant mouse CXCL11?

Proper storage of recombinant mouse CXCL11 is critical for maintaining its biological activity over time. For long-term storage, the protein should be stored at -20°C or preferably -80°C . When in liquid form, recombinant CXCL11 should be stored in Tris/PBS-based buffer containing 5-50% glycerol to prevent protein denaturation during freeze-thaw cycles . It is essential to aliquot the protein into single-use volumes to avoid repeated freeze-thaw cycles, which significantly reduce activity. Working aliquots can be stored at 4°C for up to one week, but longer storage should be at freezing temperatures . In general, protein in liquid form remains stable for up to 6 months when properly stored with glycerol at -20°C or -80°C. Lyophilized protein is more stable during shipping and storage, with the lyophilization buffer typically consisting of Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

How is recombinant mouse CXCL11 utilized in cancer immunotherapy research?

Recombinant mouse CXCL11 has emerged as a valuable tool in cancer immunotherapy research, particularly in enhancing oncolytic virus therapy. Researchers have created an oncolytic vaccinia virus expressing murine CXCL11 (vvDD-CXCL11) that selectively targets tumor cells while attracting immune cells to the tumor microenvironment . This approach leverages CXCL11's ability to specifically attract activated T (Th1) and NK cells, rather than naïve immune cells. Compared to chemokines like CCL5 that attract both naïve and activated immune cells, CXCL11 offers superior specificity for activated anti-tumor immune cells . In experimental models of mesothelioma (AB12) and colon cancer (MC38), vvDD-CXCL11 demonstrated superior therapeutic efficacy compared to control viruses. The virus efficiently replicated in tumor cells while producing approximately 20-fold more CXCL11 expression in the tumor microenvironment, enhancing immune cell infiltration and anti-tumor activity .

What correlations exist between CXCL11 expression and immune infiltration in tumors?

CXCL11 expression shows significant correlations with immune cell infiltration across multiple cancer types. Research indicates that CXCL11 expression positively correlates with CD8+ T cell and T follicular helper cell infiltration in tumors, while showing negative associations with myeloid-derived suppressor cells (MDSCs) across various cancer types . These correlations suggest a potential role for CXCL11 in shaping the tumor immune microenvironment. Additionally, CXCL11 expression correlates with mismatch repair (MMR) genes in 12 cancer types, including bladder cancer (BLCA), breast cancer (BRCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), skin cutaneous melanoma (SKCM), thymoma (THYM), and uveal melanoma (UVM) . These correlations suggest that CXCL11 may serve as a biomarker for immunotherapy response in these cancer types.

How does CXCL11 relate to established biomarkers for immunotherapy response?

CXCL11 expression shows significant relationships with established biomarkers for immunotherapy response, including mismatch repair (MMR) genes, tumor mutational burden (TMB), and microsatellite instability (MSI). MMR-deficient cancers typically have higher neoantigen loads, making them more responsive to immune checkpoint blockade therapy regardless of tissue origin . Research has demonstrated that CXCL11 expression correlates with MMR genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) across multiple cancer types . These correlations suggest that CXCL11 expression might predict responsiveness to immune checkpoint inhibitors. Additionally, CXCL11 shows positive associations with numerous immune-related genes, including other chemokines, immunostimulatory genes, and MHC genes across 33 cancer types . This extensive network of correlations positions CXCL11 as a potential comprehensive biomarker for immune activity in tumors and predictor of immunotherapy outcomes.

What factors can affect recombinant mouse CXCL11 activity in experimental settings?

Several factors can impact the activity of recombinant mouse CXCL11 in experimental settings. Improper reconstitution is a primary concern - vigorous shaking or vortexing can disrupt protein structure and significantly reduce biological activity . Buffer composition also plays a critical role, with optimal buffers typically being Tris/PBS-based with appropriate glycerol concentration . Protein concentration is another important factor; working within the recommended concentration range (0.1-1.0 mg/mL after reconstitution) ensures optimal activity . Temperature fluctuations, particularly repeated freeze-thaw cycles, can lead to protein denaturation and loss of activity . Endotoxin contamination, which should be below 0.1 EU per 1 μg, can confound experimental results by inducing inflammatory responses independent of CXCL11 activity . Finally, the target cells used in chemotaxis assays must express sufficient levels of the CXCR3 receptor to demonstrate CXCL11-mediated effects.

How can researchers validate the purity and activity of recombinant mouse CXCL11?

Validation of recombinant mouse CXCL11 should include multiple complementary approaches. SDS-PAGE analysis under reducing and non-reducing conditions can confirm protein purity (which should be >90%) and molecular weight (approximately 8.8 kDa for the core protein, or 12.9 kDa with an N-terminal His-tag) . Functional validation through chemotaxis assays using BaF3 cells transfected with human CXCR3 provides direct evidence of biological activity, with an expected ED50 below 10 ng/mL . Endotoxin testing using the LAL method ensures levels are below 0.1 EU per 1 μg of protein . For research requiring absolute certainty of protein identity, mass spectrometry can confirm the amino acid sequence. For applications involving gene therapy or oncolytic viruses expressing CXCL11, RT-qPCR and ELISA assays can verify CXCL11 expression levels in target cells or tissues . When moving to in vivo studies, validation should include assessment of immune cell infiltration to confirm the chemotactic activity of the recombinant protein.

What experimental controls should be included when working with recombinant mouse CXCL11?

When designing experiments with recombinant mouse CXCL11, several controls are essential for proper interpretation of results. For chemotaxis assays, a negative control using buffer without CXCL11 establishes baseline migration, while a positive control using an established chemotactic factor (such as CXCL10) confirms assay functionality. A dose-response curve spanning concentrations below and above the expected ED50 (typically 1-100 ng/mL) should be performed to determine optimal working concentrations . When studying CXCL11 in the context of gene therapy or oncolytic viruses, appropriate viral controls lacking CXCL11 expression (such as the parental vvDD virus in comparison to vvDD-CXCL11) are essential . For receptor-specificity studies, cells lacking CXCR3 expression should be included to confirm receptor-dependence of observed effects. In studies examining immune cell infiltration, multiple immune cell markers should be assessed to distinguish CXCL11-specific effects on CXCR3+ cells from non-specific immune responses. Finally, endotoxin-free reagents and endotoxin testing are crucial to prevent misattribution of inflammatory effects.

How does mouse CXCL11 compare structurally and functionally to human CXCL11?

Mouse CXCL11 and human CXCL11 share fundamental structural characteristics but display important species-specific differences. Both are members of the CXC chemokine family and function through binding to the CXCR3 receptor. Mouse CXCL11 has a molecular weight of 8.8 kDa and contains 79 amino acid residues, while maintaining the characteristic CXC motif essential for its chemotactic function . The amino acid sequence homology between mouse and human CXCL11 is approximately 68%, with conservation of critical functional domains. Despite these differences, both proteins demonstrate similar biological activities in their respective species, including T-cell activation and chemotaxis . When conducting cross-species studies, researchers should be aware that while mouse CXCL11 can bind to human CXCR3, the affinity and downstream signaling may differ from human CXCL11 binding to human CXCR3. These species-specific differences highlight the importance of using species-matched chemokines and receptors in preclinical research when possible.