Recombinant Human Transforming growth factor beta-1 proprotein (TGFB1), partial  (Active)

What is TGFB1 and what are its primary functions in biological systems?

TGFB1 (Transforming Growth Factor Beta 1) is a secreted 25.6 kDa homodimeric polypeptide that belongs to the TGF-beta superfamily of cytokines. It is synthesized as a preproprotein that undergoes proteolytic processing to generate a latency-associated peptide (LAP) and a mature peptide . TGFB1 functions as a multifunctional regulator involved in:

• Cell proliferation, differentiation, and growth

• Immune response modulation

• Extracellular matrix synthesis and modeling

• Wound healing and tissue repair

• Epithelial-to-mesenchymal transition

• Apoptosis regulation

TGFB1 exerts its effects by binding to type II and type I serine/threonine kinase receptors, which activate intracellular signaling primarily through SMAD proteins but also through non-canonical pathways . Its activity is context-dependent and tissue-specific, requiring precise regulation to maintain homeostasis.

How is recombinant TGFB1 typically produced for research applications?

Recombinant human TGFB1 is produced in several expression systems, with each offering different advantages:

• CHO cells: Produces glycosylated homodimeric TGFB1 with post-translational modifications similar to native protein

• HEK293 cells: Results in properly folded protein with a disulfide bond linking the two 112-amino acid subunits

• E. coli: Less commonly used due to lack of proper glycosylation

The production process typically involves:

• Gene cloning and vector construction

• Transfection into the appropriate cell line

• Protein expression under optimized conditions

• Purification via chromatographic techniques

• Quality control testing for bioactivity

For example, HEK293-derived TGFB1 demonstrates high potency with EC50 values of 1.45-1.54 pM (~36 pg/ml) in luciferase reporter assays, significantly more potent than some commercially available alternatives .

What are the optimal storage and reconstitution conditions for recombinant TGFB1?

For maximum stability and activity of recombinant TGFB1:

Storage:

• Lyophilized protein should be stored at -20°C to -80°C

• Reconstituted protein is stable for 1-2 weeks at 4°C or can be aliquoted and stored at -20°C or -80°C for longer periods

Reconstitution:

• It is recommended to reconstitute lyophilized TGFB1 in sterile 10mM HCl at a concentration of 0.1 mg/ml

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Allow protein to equilibrate to room temperature before reconstitution

• Gentle mixing rather than vortexing is recommended to avoid protein denaturation

Working solution preparation:

• The reconstituted stock can be further diluted into appropriate cell culture media or buffers

• For cell culture applications, final HCl concentration should be minimized by dilution into media containing buffering capacity

• Carrier proteins (0.1-1% BSA) may be added to prevent adhesion to tubes and improve stability in very dilute solutions

How should researchers verify the bioactivity of recombinant TGFB1 before experimental use?

Researchers should employ multiple methods to confirm TGFB1 bioactivity:

Quantitative bioassays:

• Inhibition of IL-4-induced proliferation of HT-2 cells: The ED50 of active TGFB1 should be approximately 0.0149 ng/ml, corresponding to a specific activity of 6.7×10^7 units/mg

• Induction of SMAD2/3 phosphorylation in responsive cell lines (quantified by Western blot)

• Luciferase reporter assays in transiently transfected HEK293T cells containing SMAD-responsive elements

Functional validation:

• Morphological changes in responsive cells (e.g., spheroid formation in intestinal organoids after 24-hour treatment)

• Upregulation of known TGFB1-responsive genes using qRT-PCR (e.g., SMAD7, PAI-1, or COL1A1)

• Induction of epithelial-to-mesenchymal transition markers in appropriate cell types

Quality control benchmarks:

• Purity >95% by SDS-PAGE and HPLC

• Endotoxin levels <1 EU/μg protein for cell culture applications

• Correct molecular weight verification by mass spectrometry

How does TGFB1 expression correlate with cancer progression and prognosis?

TGFB1 exhibits complex, context-dependent roles in cancer, which vary by cancer type and stage:

Expression patterns and correlations:

• TGFB1 is broadly dysregulated in hematological malignancies and generally associated with adverse prognosis

• In colorectal cancer, higher relative expression is associated with lack of vascular invasion by cancer cells and presence of lymphocytes in neoplastic tissue

• TGFB1 shows a hematologic-tissue-specific expression pattern both across normal tissues and cancer types

• In AML, high TGFB1 expression correlates with specific genetic mutations (more frequent NRAS and DNMT3A mutations, less frequent WT1 mutation)

Prognostic value:

How can researchers effectively analyze TGFB1's effects on immune cell infiltration and the tumor microenvironment?

Researchers should utilize multiple complementary approaches:

Computational methods:

Experimental methods:

• Single-cell RNA sequencing to analyze TGFB1 expression at cellular resolution

• Co-culture experiments with TGFB1-treated cells and immune cell populations

• Flow cytometry to quantify changes in immune cell populations after TGFB1 treatment

• Immunohistochemistry to visualize immune cell infiltration in tissue samples

Key findings to validate:

• TGFB1 expression positively correlates with macrophages and monocytes infiltration in most cancers

• TGFB1 expression shows positive correlation with CD8+ T cells and NK cells but negative correlation with CD4+ T cells

• TGFB1 expression is positively correlated with stromal scores in multiple hematological malignancies including AML, CHL, CLL, CML, DLBCL, MCL, MDS, MM, pre-B ALL, and T-ALL

What are the optimal dosing and timing considerations when using recombinant TGFB1 in cell culture experiments?

Effective TGFB1 treatment protocols depend on the experimental goals and cell types:

Dosing recommendations:

• For inducing regenerative responses in intestinal organoids: 4-24 hour exposure at 5-20 ng/ml

• For activating SMAD signaling in most cell lines: 1-10 ng/ml

• For mesenchymal cell stimulation: Dose-dependent response observed between 0.1-10 ng/ml, with optimal clustering observed at higher concentrations

• For immune cell modulation: Generally 1-5 ng/ml, though regulatory T cells may require different concentrations

Timing considerations:

• Acute responses (SMAD phosphorylation): 30 minutes to 2 hours

• Gene expression changes: 4-24 hours

• Morphological changes: 24-72 hours

• Long-term effects (differentiation, EMT): 3-14 days with repeated treatments

Experimental design strategies:

• Include time-course experiments to capture both early and late TGFB1-mediated effects

• For regeneration studies, a single 24-hour dose of TGFB1 following irradiation (4 Gy) can induce spheroid morphology and elevation in fetal and regenerative cell transcripts in intestinal organoids

• Pre-treatment of mesenchymal cultures with TGFB1 before co-culture with epithelial cells shows dose-dependent induction of regenerative transcripts in the epithelium

How can researchers effectively study TGFB1 secretion and activation mechanisms?

Investigating TGFB1 secretion and activation requires specialized techniques:

For studying unconventional secretion pathways:

• Co-immunoprecipitation to detect TGFB1 interaction with secretory pathway components like GORASP2/GRASP55

• Live-cell imaging with fluorescently tagged TGFB1 to track secretory vesicles

• Genetic manipulation of autophagy components (ATG proteins) to assess their role in TGFB1 secretion

• Analysis of RAB8A-dependent pathways using dominant-negative constructs or siRNA

For investigating TGFB1 activation:

• Bioassays comparing active versus total TGFB1 (with and without acid activation)

• Analysis of integrin-mediated activation using inhibitors of αV integrins (ITGAV:ITGB6 or ITGAV:ITGB8)

• Studying the role of proteases in TGFB1 activation using specific inhibitors

• Examination of LAP interaction with matrix components through solid-phase binding assays

Molecular tools:

• CRISPR/Cas9 to delete components of the TGFB1 secretory or activation machinery

• Fluorescent reporters to monitor spatial and temporal aspects of TGFB1 secretion

• Antibodies that specifically detect latent versus active TGFB1

• Co-culture systems to study cell-cell communication in TGFB1 activation

How can researchers address variability in TGFB1 responsiveness across different cell lines and experimental systems?

Variability in TGFB1 responses is a significant challenge that can be managed through:

Characterization of cellular context:

• Measure baseline expression of TGFB receptors (TGFBR1, TGFBR2) and downstream signaling components

• Assess endogenous production of TGFB family members that might compete for receptor binding

• Evaluate expression of TGFB1 inhibitors like decorin or specific antagonists

• Determine the methylation status of the TGFB1 promoter region, which can influence expression levels

Technical considerations:

• Use multiple readouts for TGFB1 activity (phospho-SMAD Western blots, target gene qPCR, phenotypic changes)

• Include positive control cell lines with well-characterized TGFB1 responses

• Standardize culture conditions, as serum components can contain TGFB1 or TGFB1 inhibitors

• Consider species differences in TGFB1 responses, as studies have shown differential responses between rat and mouse microglia

Experimental design strategies:

• Perform dose-response curves for each new cell line or primary culture

• Use animal-free recombinant TGFB1 for more reproducible results in chemically-defined media

• Establish internal laboratory standards and positive controls for normalization across experiments

• Document passage number and culture history as these can affect TGFB1 responsiveness

What are the methodological considerations for analyzing TGFB1 gene expression in tissue samples?

Accurate analysis of TGFB1 gene expression requires careful attention to:

Sample preparation:

• Rapid tissue processing and RNA extraction to prevent degradation

• DNase treatment to eliminate genomic DNA contamination

• Assessment of RNA quality using bioanalyzer or gel electrophoresis

• Consistent tissue sampling techniques to account for heterogeneity

qRT-PCR methodology:

• Selection of appropriate reference genes (e.g., GAPDH) with verified stability across experimental conditions

• Calculation of PCR efficiency using standard curves (e.g., 101% for TGFB1 and 111% for GAPDH as reported)

• Use of the Pfaffl's method to calculate relative expression ratio when PCR efficiencies differ between target and reference genes

• Amplification in triplicate with negative controls to ensure reliability

Data analysis considerations:

• Statistical comparison with clinico-pathological features requires substantial sample sizes (e.g., n=64 in colorectal cancer studies)

• Correlation with methylation status of the promoter region (-235 to +22 nucleotide from transcription start)

• Stratification by disease stage, histological subtype, or other relevant clinical parameters

• Use of appropriate statistical tests (e.g., STATISTICA software for evaluating correlations)

How is TGFB1 being explored as a biomarker or therapeutic target in immunotherapy research?

TGFB1 shows significant promise in immunotherapy applications:

As a biomarker:

As a therapeutic target:

• TGFβ1-inhibitory therapies may restore cancer immunity and synergize with other immunotherapies

• Inhibition of TGFB1 signaling has been shown to synergize with anti-PD-L1 therapy in experimental models

• TGFB1 inhibition facilitates T-cell penetration and improves outcomes of immunotherapy

• Modulating TGFB1 signaling shows promise for improving ineffective erythropoiesis in myelodysplastic syndrome

Methodological approaches:

• Development of TGFβ1-specific gene signatures to predict immunotherapy responses

• Combined assessment of mRNA and protein aspects of TGFB1 in prospective immunotherapy studies

• Analysis of TGFB1 signaling in specific cell populations within the tumor microenvironment

What are emerging techniques for studying TGFB1's role in cellular reprogramming and regeneration?

Cutting-edge approaches for studying TGFB1 in regeneration include:

Advanced model systems:

• Intestinal organoid cultures treated with TGFB1 to study fetal-like regenerative states

• Co-culture systems with epithelial and mesenchymal components to study cell-cell communication

• In vivo models of tissue damage followed by TGFB1 administration or inhibition

• Pre-treatment of primary epithelial cultures with TGFB1 to enhance engraftment into damaged tissues

Multi-omics approaches:

• Integration of ATAC-seq, scRNA-seq, and ChIP-seq to identify regenerative transcriptional circuits activated by TGFB1

• Time-series experiments to capture the dynamic nature of TGFB1-induced regenerative responses

• Spatial transcriptomics to map TGFB1 signaling within tissue architecture

• Proteomics to identify post-translational modifications induced by TGFB1

Key research findings:

• TGFB1 activates YAP-SOX9 transcriptional circuits in epithelium, promoting regeneration

• TGFB1 signaling is activated in the intestine post-irradiation, with monocytes/macrophages being the main source

• Pre-treatment with TGFB1 enhances the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting therapeutic potential

• TGFB1 reshapes mesenchymal signaling environment to favor regenerative growth by increasing levels of regeneration-promoting factors (Ptgs2, Wnt5a, Lif) while decreasing homeostatic growth factors (Grem1, Rspo3)