TXN1, His
Description
Production and Biochemical Characteristics
TXN1, His is produced recombinantly in E. coli as a single polypeptide chain (117 amino acids, residues 2–109) with a molecular mass of 12.8 kDa . Key features include:
| Parameter | Description |
|---|---|
| Expression System | E. coli |
| Tag | N-terminal 9-amino acid His tag (for affinity purification) |
| Purity | >95% (SDS-PAGE) |
| Formulation | 20 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM DTT |
| Storage | 4°C (short-term), -20°C (long-term) |
The protein is purified using proprietary chromatographic techniques and retains redox activity, as evidenced by its ability to reduce insulin, though with distinct kinetics compared to native TXN1 .
Core Structure
TXN1, His retains the canonical thioredoxin fold, characterized by:
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Active Site Motif: -Cys32-Gly-Pro-Cys35- (reduces disulfide bonds via dithiol-disulfide exchange) .
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Additional Cysteines: Mammalian TXN1 contains Cys62, Cys69, and Cys73, which undergo post-translational modifications (e.g., S-nitrosylation, glutationylation) under oxidative stress but do not contribute to redox activity .
Functional Roles
TXN1, His participates in redox-dependent and -independent pathways:
Research Applications
TXN1, His is widely used to study:
Hematopoietic Stem Cell (HSC) Biology
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Radiation Protection: Recombinant TXN1, His administration rescues mice from lethal radiation by suppressing TP53 signaling and promoting HSC proliferation .
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Transplantation: Enhances hematopoietic recovery in allogeneic stem cell transplants by mitigating oxidative stress .
Inflammation and Immunity
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Macrophage Polarization: Supports M1 macrophage activation (e.g., Nos2 expression) via TrxR1-dependent pathways, linking redox balance to pro-inflammatory responses .
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Monocyte Survival: Maintains NF-κB activity in monocytes and dendritic cells, enabling pathogen responses .
Cardiac and Metabolic Regulation
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AMPK Stabilization: Forms disulfide bonds with AMPK, preventing aggregation during oxidative stress and preserving energy metabolism .
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Hypertrophy Suppression: Reduces HDAC4, promoting nuclear import and inhibition of NFAT-driven hypertrophic genes .
Comparative Analysis with Native TXN1
Clinical and Experimental Challenges
Product Specs
Q&A
What is TXN1 and what are its primary cellular functions?
TXN1 (Thioredoxin-1) is one of the major cellular antioxidant proteins in mammals, belonging to the thioredoxin system that includes thioredoxin reductase, thioredoxin, and NADPH as an electron donor. Beyond its direct antioxidant functions, TXN1 is involved in a wide range of physiological cellular responses both dependent and independent of reactive oxygen species (ROS) . The protein maintains reducing power for ribonucleotide reductase, which is essential for DNA replication and repair . TXN1 is produced by multiple cell types including hepatocytes, fibroblasts, activated monocytes, and lymphocytes, and can function in a chemokine-like manner to induce cell migration and proliferation .
How does TXN1 interact with the TP53 pathway?
TXN1 regulates the TP53 tumor suppressor pathway, forming a TXN1-TP53 axis that is crucial for hematopoietic stem/progenitor cell (HSPC) biological fitness. Deletion of TXN1 in HSPCs activates the TP53 signaling pathway and attenuates HSPC capacity to reconstitute hematopoiesis . This regulatory interaction provides an attractive alternative approach to directly targeting TP53 for enhancing stem cell function in hematopoietic stem cell transplantation (HSCT) and in radiation injury . The relationship between these two proteins represents a central regulatory mechanism in HSPC biological functions.
What phenotypes are observed in TXN1 knockout or mutation models?
TXN1 knockout models display distinct phenotypes depending on the specific tissues affected:
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Constitutive homozygous deletion of TXN1 is embryonically lethal, necessitating conditional knockout approaches for research
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In ROSA-CreER-TXN1 fl/fl mice, tamoxifen-induced TXN1 deletion impairs HSPC function
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Rats with TXN1-F54L mutation show vacuolar degeneration in the midbrain, particularly in the thalamus and inferior colliculus, with neuronal and oligodendrocyte cell death
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TXN1-F54L rats exhibit morphological changes in mitochondria and significantly reduced TXN1 protein levels (approximately one-third of wild-type)
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In these rat models, vacuolar degeneration begins at three weeks of age with spontaneous repair beginning at seven weeks
What are effective approaches for generating TXN1 knockout models?
For conditional TXN1 knockout models, multiple approaches have proven successful:
How can researchers assess TXN1 function in hematopoietic stem cells?
Assessment of TXN1 function in hematopoietic stem cells (HSCs) requires multiple complementary approaches:
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Flow Cytometry Analysis: To enumerate and characterize HSPC populations
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Limiting Dilution Competitive Transplantation: With sorted HSCs to assess self-renewal
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Serial Transplantations: To evaluate long-term reconstitutional capacity
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RNA Sequencing: To investigate downstream molecular pathways
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Colony Forming Assays: To measure progenitor function and differentiation potential
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Radiation Sensitivity Testing: To evaluate TXN1's role in radiation protection
For transplantation experiments specifically, researchers should consider both primary and secondary transplantation to fully assess HSC self-renewal, with flow cytometric analysis of peripheral blood at 4, 8, 12, and 16 weeks post-transplantation for comprehensive evaluation of reconstitution potential .
What molecular techniques are most effective for studying TXN1-regulated pathways?
To elucidate TXN1-regulated pathways, researchers can employ these validated approaches:
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ChIP-PCR: To identify TXN1-associated transcription factors and their genomic targets
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PGL3 Reporter Assays: To study transcriptional regulation mechanisms
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TP53 Protein Degradation Assays: To examine how TXN1 influences TP53 stability
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CRISPR/Cas9 Knockout: In cell lines (such as EML murine hematopoietic stem/progenitor cell line) for mechanistic studies
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Western Blotting: To quantify protein expression levels in different tissues
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qPCR: To validate expression changes of key pathway components
When analyzing TXN1-TP53 pathway interactions specifically, it is advisable to assess multiple components of the pathway including downstream targets like p21, PUMA, and BAX to comprehensively understand regulatory mechanisms .
How does TXN1 coordinate with other antioxidant systems?
TXN1 functions as part of an interconnected network of antioxidant systems. When TXN1 or Thioredoxin Reductase-1 (Txnrd1) is depleted, cells activate compensatory mechanisms:
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Nrf2 Pathway Activation: Deletion of Txnrd1 leads to constitutive stability of Nrf2, a master regulator of antioxidant responses
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Glutathione System Upregulation: Txnrd1-deficient cells show increased expression of glutathione synthesis genes (Slc7a11, Gclc) and glutathione transferases (Gstm1, Gstm2, Gsto1, Gstp1, Gstp2)
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Heme Metabolism: Altered expression of genes involved in heme binding (Hebp1) and iron homeostasis (Ftl1, Meltf, Steap4)
Research shows that Txnrd1-deficient β-cells have increased total glutathione levels compared to controls, suggesting a metabolic shift from Trx/Prx-based defense to mechanisms relying on glutathione and biliverdin/bilirubin pathways . This compensation explains why acute depletion of TXN1 components may produce different phenotypes than genetic knockout models where adaptive responses have time to develop.
What is the specific role of TXN1 in DNA synthesis and cell proliferation?
TXN1 plays a crucial role in DNA synthesis through multiple mechanisms:
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Ribonucleotide Reductase Support: TXN1 is the exclusive protein maintaining reducing power for ribonucleotide reductase, the essential enzyme for DNA building blocks
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2'-Deoxyribonucleotide Provision: The Trx1 system is essential for the final step of nucleotide biosynthesis
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DNA Damage Prevention: Impaired availability of 2'-deoxyribonucleotides due to TXN1 deficiency induces DNA damage response and cell cycle arrest
In T-cell development and activation specifically, c-Myc-dependent activation of the Trx1 system is critical during proliferation, while the system is repressed during T-cell quiescence . Deletion of Txnrd1 prevents expansion of the CD4-CD8- thymocyte population and impairs T-cell expansion during viral and parasite infection . This essential role in DNA synthesis makes targeting Txnrd1, rather than TXN1 directly, a potential strategy for treating T-cell leukemia .
How can researchers address potential compensatory mechanisms when studying TXN1 deletion?
When designing experiments to study TXN1 deletion effects, researchers should:
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Compare Acute vs. Chronic Depletion: Utilize both inducible knockout systems and acute inhibition approaches to distinguish immediate effects from compensatory adaptations
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Analyze Multiple Antioxidant Pathways: Measure changes in glutathione system components, Nrf2 pathway activation, and heme metabolism genes
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Include Time-Course Analyses: Evaluate phenotypes at different time points post-deletion to capture dynamic compensatory responses
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Combine Genetic and Pharmacological Approaches: Use TXN1 inhibitors alongside genetic models to confirm phenotypes
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Monitor Nrf2 Activation: Assess nuclear localization of Nrf2 and expression of its target genes as indicators of compensatory responses
Studies have demonstrated that β-cells respond to Txnrd1 loss by stabilizing Nrf2, increasing expression of genes involved in heme- or glutathione-based antioxidant mechanisms . Similar Nrf2 stabilization has been observed in Txnrd1-deficient mouse hepatocytes , suggesting this is a conserved compensatory response.
What are the best experimental models for studying TXN1 in radiation protection?
To investigate TXN1's radioprotective effects, researchers have successfully employed:
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Myeloablative HSCT Mouse Models: TXN1 is significantly upregulated in bone marrow of HSCT recipient mice treated with AMD3100 (plerixafor)
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Ex-vivo HSC Culture: Culture with recombinant TXN1 enhances HSC long-term repopulation capacity
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Total Body Irradiation Models: Administration of recombinant TXN1 up to 24 hours following lethal TBI rescues BALB/c and C57Bl/6 mice from radiation-induced lethality
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In Vitro Radiation Sensitivity Assays: TXN1 knockout renders HSPCs more sensitive to radiation, while recombinant TXN1 promotes HSPC proliferation and expansion
When designing radiation protection studies, it is critical to establish proper timing of TXN1 administration relative to radiation exposure, as the effective window (up to 24 hours post-radiation) has been experimentally determined . Additionally, investigators should consider both TXN1's direct antioxidant effects and its potential influence on DNA repair pathways.
What are promising translational applications of TXN1 research findings?
Based on current research, several translational applications of TXN1 research show promise:
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Hematopoietic Recovery Enhancement: Recombinant TXN1 has demonstrated potential for enhancing recovery following myeloablative hematopoietic stem cell transplantation
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Radiation Countermeasures: TXN1 administration up to 24 hours post-radiation exposure could serve as a medical countermeasure for radiation accidents or incidents
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Targeting TXN1-TP53 Axis: Modulating this axis offers an alternative approach to enhancing stem cell function rather than directly targeting TP53
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T-cell Leukemia Treatment: Targeting Txnrd1 may provide a therapeutic strategy for T-cell leukemia based on its role in DNA synthesis and proliferation
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Neurological Disorder Therapies: Understanding TXN1's role in midbrain development could inform approaches to neurological conditions
Future studies should focus on defining optimal dosing regimens for recombinant TXN1, identifying small molecule enhancers of TXN1 activity, and developing targeted delivery approaches to maximize therapeutic efficacy while minimizing potential systemic effects.
How might single-cell approaches advance our understanding of TXN1 biology?
Single-cell technologies offer several advantages for TXN1 research:
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Heterogeneity Assessment: Single-cell RNA sequencing can reveal differential TXN1 expression and pathway activation across subpopulations of cells
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Temporal Dynamics: Single-cell trajectory analysis can map how TXN1-related pathways change during developmental processes or stress responses
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Niche Interactions: Spatial transcriptomics can uncover how TXN1 expression in specific cells influences neighboring populations
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Compensatory Mechanism Resolution: Single-cell approaches can distinguish between cell populations that successfully compensate for TXN1 loss versus those that fail to adapt
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Redox State Monitoring: Integration of single-cell proteomics with redox sensors could provide unprecedented insight into TXN1's influence on cellular redox states These approaches would be particularly valuable for understanding the variable sensitivity of different HSPC subpopulations to TXN1 manipulation, potentially identifying specific cellular contexts where TXN1 intervention would be most beneficial.
Product Science Overview
Thioredoxin exists as a disulfide-linked homodimer and contains a single thioredoxin domain . The protein participates in redox reactions through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol-disulfide exchange reactions . This activity is essential for maintaining the redox balance within cells and protecting against oxidative damage.
Recombinant thioredoxin is produced using various expression systems, with E. coli being one of the most common hosts . The recombinant protein is often tagged with a His tag (histidine tag) to facilitate purification. The His tag is a sequence of histidine residues that binds strongly to nickel ions, allowing for easy isolation of the protein using nickel-affinity chromatography .
Recombinant thioredoxin, especially with a His tag, is widely used in research and biotechnology. Some of its applications include:
- Protein Folding: Thioredoxin assists in the correct folding of proteins by catalyzing the formation and isomerization of disulfide bonds.
- Redox Studies: It is used to study redox reactions and signaling pathways within cells.
- Fusion Protein: Thioredoxin can be fused to other proteins to enhance their solubility and stability during expression and purification .
Recombinant thioredoxin is typically provided as a lyophilized powder and should be stored at -20°C to -80°C for long-term stability . Once reconstituted, the protein solution can be stored at 4-8°C for short-term use. Proper storage conditions are crucial to maintain the protein’s activity and prevent degradation .
