TXN1 E.Coli
Description
Table 1: Molecular Properties of TXN1 Variants
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Active Site: Contains a conserved Cys-Gly-Pro-Cys motif enabling redox activity via reversible disulfide bond formation .
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Structure: Comprises two conformational domains (β-α-β-α-β and β-β-α) stabilized by hydrophobic clusters and a central β-sheet .
Biological Functions and Mechanisms
TXN1 E.Coli serves as a general protein disulfide oxidoreductase with roles in:
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Redox Regulation: Catalyzes dithiol-disulfide exchange reactions, critical for reducing ribosomal proteins, transcription factors, and enzymes like ribonucleotide reductase .
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Protein Folding: Assists in forming correct disulfide bonds during recombinant protein production in E. coli, enhancing solubility and yield of antibodies and other therapeutics .
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DNA Synthesis: Directly supports ribonucleotide reductase activity, enabling deoxyribonucleotide production .
Applications in Biotechnology
TXN1 E.Coli is integral to:
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Recombinant Protein Production: Enhances solubility and correct folding of disulfide-rich proteins .
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Antibody Engineering: Fusion with TXN1 improves cytoplasmic expression of scFv antibodies in E. coli, bypassing periplasmic secretion requirements .
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Redox Assays: Used to monitor insulin reduction kinetics, with activity quantified by absorbance changes at 650 nm .
Recent Research Advances
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NF-κB Activation: TXN1 facilitates NF-κB DNA binding in immune cells, linking redox signaling to inflammatory responses .
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Oxidative Stress Response: TXN1-deficient E. coli strains show heightened sensitivity to diamide and ferric iron, underscoring its role in oxidative defense .
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Enzyme Engineering: Truncated TXN1 variants retain redox activity, enabling tailored applications in biocatalysis .
Product Specs
Q&A
What is thioredoxin (TXN1) and what is its role in E. coli?
Thioredoxin (TXN1) is a small redox-active protein approximately 12 kDa in size that is ubiquitously found in E. coli cells. It functions primarily as a disulfide reductase through its conserved active site cysteines, participating in critical cellular processes including DNA replication, sulfur metabolism, and oxidative stress response. E. coli B strains contain approximately 10,000 copies of thioredoxin per cell, with the majority located in the soluble fraction after membrane separation through gentle lysis and centrifugation . The protein is essential for certain phage replications, particularly phage T7 DNA replication, and plays a significant role in the biosynthesis of L-cystine through enzymatic reduction of sulfate .
How is thioredoxin measured or quantified in E. coli samples?
Researchers can quantify thioredoxin in E. coli using several complementary methods:
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Radioimmunoassay: A competition radioimmunoassay using Y-labeled thioredoxin-S and a double antibody technique can detect picomole amounts of thioredoxin in crude cell extracts .
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Enzymatic assays: These employ excess thioredoxin reductase and NADPH in the reduction of:
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Immunological methods:
These methods can be used to determine thioredoxin levels under various experimental conditions and in different cellular compartments.
What are common E. coli strains used for thioredoxin research?
Several E. coli strains are particularly valuable for thioredoxin research:
| Strain | Characteristics | Research Applications |
|---|---|---|
| E. coli B | Wild-type strain with ~10,000 copies of thioredoxin per cell | Baseline studies, protein localization |
| E. coli B tsnC mutants | Defective in phage T7 DNA replication | Studying thioredoxin's role in DNA replication |
| E. coli B tsnC 7004 | Contains no detectable thioredoxin | Negative control, studying cellular effects of thioredoxin absence |
| E. coli trxB gor | Double mutant in glutathione oxidoreductase (gor) and thioredoxin reductase (trxB) | Enhanced disulfide bond formation in cytoplasm |
| E. coli trxB | Single mutant in thioredoxin reductase | Studying redox pathways |
The E. coli B tsnC 7004 strain is particularly valuable as it appears to be a nonsense or deletion mutant, showing no detectable levels of thioredoxin across multiple detection methods .
How can thioredoxin fusion proteins be used to enhance protein expression in E. coli?
Thioredoxin fusion proteins significantly enhance the expression and correct folding of heterologous proteins in E. coli cytoplasm, particularly for proteins requiring disulfide bonds for proper folding. When designing thioredoxin fusion constructs:
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Position thioredoxin at the N-terminus of the target protein.
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Include an appropriate linker sequence between thioredoxin and the target protein.
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Consider using E. coli trxB gor double mutant strains that enhance disulfide bond formation in the cytoplasm .
Thioredoxin fusions have demonstrated superior results compared to other fusion partners (such as maltose-binding protein) for enhancing scFv antibody expression, yielding higher expression levels and better folding outcomes, even without co-expression of chaperones in some cases .
Data shows that Trx1-scFv fusions produce correctly folded proteins in the E. coli trxB gor cytoplasm, with properly formed disulfide bridges, which doesn't occur in trxB single mutants. Importantly, the Trx1 fusion has minimal impact on the affinity of the scFv, meaning the fusion protein can often be used without removing the Trx1 portion .
What growth conditions optimize thioredoxin expression and function in E. coli?
Optimal growth conditions for thioredoxin expression and function in E. coli include:
For strains deficient in thioredoxin (such as E. coli B tsnC 7004), supplementation with L-cystine normalizes growth behavior, supporting evidence that thioredoxin contributes to L-cystine biosynthesis through sulfate reduction pathways .
What are effective methods for analyzing thioredoxin-protein interactions in E. coli?
Several methods can be employed to analyze thioredoxin-protein interactions in E. coli:
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Conjugation assays: Can be quantified using time-to-threshold methods where cell mixtures are diluted in media with dual antibiotic selection and OD₆₀₀ is monitored to determine conjugation efficiency .
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Antigen-binding assays: These are useful for assessing the functional activity of thioredoxin fusion proteins, particularly antibody fragments. They can determine whether the thioredoxin fusion affects the binding affinity of the partner protein .
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Disulfide-bond formation analysis: Can be used to assess whether thioredoxin is acting as a disulfide catalyst or as a protein chaperone in fusion proteins .
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Solubility testing: Comparing the solubility of proteins with and without thioredoxin fusion to determine the chaperone effect of thioredoxin .
How does thioredoxin function differ between wild-type and mutant E. coli strains?
Thioredoxin function shows significant differences across E. coli strains:
| Strain | Thioredoxin Characteristics | Functional Impact |
|---|---|---|
| Wild-type E. coli B | Normal levels (~10,000 copies/cell), primarily in soluble fraction | Normal growth, phage T7 DNA replication, sulfur metabolism |
| E. coli B tsnC 7004 | No detectable thioredoxin | Defective phage T7 DNA replication, slow growth in minimal media, normal growth with L-cystine supplementation |
| E. coli trxB gor | Modified redox environment due to mutations in glutathione oxidoreductase (gor) and thioredoxin reductase (trxB) | Enhanced disulfide bond formation in cytoplasm, supports correct folding of Trx1-scFv fusions |
| E. coli trxB | Single mutation affects thioredoxin reduction | Does not support correct disulfide bridge formation in Trx1-scFv fusions |
What are the mechanistic differences between thioredoxin acting as a disulfide bond catalyst versus a molecular chaperone?
Research reveals that thioredoxin serves dual roles that can be distinguished:
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As a disulfide bond catalyst:
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Requires intact catalytic cysteine residues for redox activity
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Facilitates the formation, reduction, or isomerization of disulfide bonds
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Functions through thiol-disulfide exchange reactions
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As a molecular chaperone:
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Acts independently of its redox activity
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Facilitates proper protein folding by preventing aggregation
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Can function even with mutated catalytic cysteines
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Experiments with a Trx1"AGPA" variant, where catalytic cysteine residues were mutated to alanine, demonstrated that this redox-inactive variant remained fully capable of assisting proper folding of fused scFv antibodies. This evidence supports that thioredoxin primarily acts as an intramolecular protein chaperone rather than as a disulfide bond catalyst when used as a fusion partner in the E. coli cytoplasm .
How can high-throughput techniques be applied to study thioredoxin regulation and function in E. coli?
Modern high-throughput technologies offer powerful approaches to study thioredoxin in E. coli:
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ChIP-seq/ChIP-exo: Can identify transcription factor binding sites affecting thioredoxin gene expression under various conditions .
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RNA-seq: Enables comprehensive analysis of transcriptional changes in thioredoxin and related genes across different growth conditions and genetic backgrounds .
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gSELEX and biotin-modified DAP-seq: These techniques can identify DNA-protein interactions affecting thioredoxin regulation .
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Normalized data collection: For meaningful comparative analysis, data should be uniformized and normalized using computational methods to reduce methodological differences or batch effects .
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Metadata annotation: Growth conditions and genetic backgrounds should be carefully documented using ontologies like the Microbial Conditions Ontology (MCO) to enable proper contextualization of experimental results .
What are common pitfalls when working with thioredoxin in E. coli and how can they be addressed?
Several challenges may arise when working with thioredoxin in E. coli:
When studying thioredoxin mutants, it's important to recognize that some mutations may affect cell growth differently depending on media composition. For example, E. coli B tsnC 7004 grows poorly in minimal medium but this phenotype can be rescued by L-cystine supplementation .
How should contradictory data on thioredoxin function in different E. coli strains be reconciled?
When faced with contradictory data on thioredoxin function:
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Consider strain differences: Compare the genetic backgrounds of the E. coli strains used (e.g., B strains vs. K-12 derivatives).
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Evaluate experimental conditions: Different media compositions, growth phases, and stress conditions can dramatically affect thioredoxin function and expression.
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Assess measurement techniques: Different detection methods (radioimmunoassay, enzymatic assays, immunological methods) have different sensitivities and limitations .
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Examine redox environment: The cytoplasmic redox state differs significantly between wild-type and trxB/gor mutant strains, affecting disulfide bond formation .
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Analyze specific mutations: Determine whether contradictions arise from different mutations in thioredoxin or related systems.
Research shows that thioredoxin's role can vary significantly depending on these factors, with some functions being essential only under specific conditions (such as minimal media growth) but dispensable under others (such as rich media growth) .
What emerging technologies might enhance our understanding of thioredoxin in E. coli?
Several cutting-edge approaches promise to advance thioredoxin research:
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CRISPR-Cas9 genome editing: Enables precise manipulation of thioredoxin and related genes to create new mutants with specific properties.
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Single-cell technologies: Allow investigation of cell-to-cell variation in thioredoxin expression and function.
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Cryo-EM and advanced structural analysis: Provide detailed insights into thioredoxin interaction with partner proteins.
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Improved computational models: Help predict thioredoxin behavior in complex cellular environments.
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Integrated multi-omics approaches: Combine transcriptomics, proteomics, and metabolomics to understand thioredoxin's role in cellular networks .
How might thioredoxin research in E. coli inform broader applications in protein engineering?
Thioredoxin research in E. coli has significant implications for protein engineering:
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Enhanced expression systems: The demonstrated success of thioredoxin fusions for improving scFv antibody production suggests broader applications for difficult-to-express proteins .
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Engineered redox environments: Understanding how mutations in the glutathione oxidoreductase (gor) and thioredoxin reductase (trxB) pathways enable disulfide bond formation in the cytoplasm provides a foundation for creating optimized expression systems .
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Chaperone engineering: The finding that thioredoxin acts primarily as a molecular chaperone even with mutated catalytic cysteines opens possibilities for designing specialized chaperones for specific protein classes .
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Synthetic biology applications: Thioredoxin components could be integrated into synthetic biological circuits designed for specific sensing or production functions.
Research demonstrating that thioredoxin's chaperone function can be separated from its redox activity (as shown with the Trx1"AGPA" variant) provides particular promise for developing specialized fusion partners optimized for specific protein production challenges .
Product Science Overview
Thioredoxin is a small, ubiquitous protein that plays a crucial role in various cellular processes by acting as a general disulfide reductant. It is found in many organisms, including bacteria, plants, and animals. In Escherichia coli, thioredoxin is involved in several essential biochemical pathways, including ribonucleotide reduction, assimilatory sulfate reduction, and the regulation of protein sulfhydryl groups .
Thioredoxin from Escherichia coli is a protein with two redox-active half-cysteine residues. These residues are critical for its function as a disulfide reductant. The protein works efficiently on model compounds and protein disulfides, facilitating the reduction of disulfide bonds in various substrates . This reduction is essential for maintaining the redox balance within the cell, which is crucial for proper cellular function and survival .
Recombinant expression of thioredoxin in E. coli has been widely used in research and biotechnology. E. coli is a popular host for the production of heterologous proteins due to its ease of genetic manipulation and rapid growth. Recombinant thioredoxin produced in E. coli has been utilized for various purposes, including the study of protein tertiary structure, structure-function experiments, enzymology, and as a bio-pharmaceutical .
However, the production of recombinant proteins in E. coli is not without challenges. One common issue is the formation of insoluble, misfolded cytoplasmic complexes known as inclusion bodies. The likelihood of inclusion body formation increases with the size and complexity of the protein . Additionally, recombinant proteins produced in E. coli may retain the N-terminal initiator methionine residue, which can affect their function and stability .
Thioredoxin has several important applications in research and biotechnology. It is used as a fusion tag to enhance the solubility and stability of recombinant proteins expressed in E. coli. This is particularly useful for proteins that are prone to forming inclusion bodies. By fusing these proteins with thioredoxin, researchers can improve their solubility and facilitate their purification .
In addition to its role as a fusion tag, thioredoxin is also involved in various cellular processes. It is required for filamentous phage assembly in vivo and catalyzes the refolding of various proteins . The thioredoxin system, which includes thioredoxin and thioredoxin reductase, is essential for maintaining the redox balance within cells. Disruption of this balance can lead to cell death and is implicated in various diseases .
