Recombinant Human Transforming growth factor beta-2 proprotein (TGFB2), partial (Active)

What is the structure of recombinant human TGF-beta 2 and how does it differ from other TGF-beta family members?

Recombinant human TGF-beta 2 belongs to the TGF-beta superfamily and exhibits a characteristic cysteine knot structure that is critical for its biological activity. The mature TGF-beta 2 protein exists as a disulfide-linked homodimer with a molecular mass of approximately 12.7 kDa, appearing as a single band at 12 kDa under reducing conditions and 24 kDa under non-reducing conditions when analyzed by SDS-PAGE . Human TGF-beta 2 is encoded by a cDNA that produces a 414 amino acid precursor containing a 19 amino acid signal peptide and a 395 amino acid proprotein that undergoes processing by furin-like convertases . The mature form used in most research applications corresponds to the C-terminal 112 amino acid segment, typically the region from Ala303 to Ser414 in the recombinant protein . Unlike TGF-beta 1 and TGF-beta 3, TGF-beta 2 has unique receptor binding properties and demonstrates non-redundant functions in development, as evidenced by knockout mouse models showing specific defects in cardiac, lung, craniofacial, and other organ systems .

What cellular processes are regulated by TGF-beta 2 in experimental models?

TGF-beta 2 functions as a cellular switch regulating multiple biological processes including immune function, cell proliferation, differentiation, and tissue remodeling. In cell culture systems, TGF-beta 2 has been demonstrated to inhibit interleukin-4-induced cell proliferation in the HT-2 mouse T cell line with an ED50 of 0.025-0.25 ng/mL, making this a useful bioassay for confirming protein activity . TGF-beta 2 plays crucial roles in regulating extracellular matrix production and remodeling, with experimental evidence showing its ability to induce fibrotic gene expression in cholangiocytes and hepatic stellate cells . Research using cranial neural crest-derived osteoprogenitor cells has revealed that TGF-beta 2 regulates the basal transcriptional machinery controlling both cell proliferation and differentiation processes . Additionally, TGF-beta 2 has been observed to exert neuroprotective effects, as it can attenuate injury-induced death of mature motoneurons in appropriate experimental contexts .

How is recombinant TGF-beta 2 produced and what quality control measures should researchers consider?

Recombinant human TGF-beta 2 for research applications is predominantly produced using mammalian expression systems, which ensure proper protein folding and post-translational modifications essential for biological activity . The recombinant protein typically expresses the region encoding Ala303-Ser414, which corresponds to the mature, active form of the cytokine . When evaluating recombinant TGF-beta 2 for experimental use, researchers should verify several quality parameters: purity (>95% by reducing SDS-PAGE), endotoxin levels (<0.001 ng/μg or 0.01 EU/μg as determined by LAL test), and biological activity through functional assays such as cell proliferation inhibition tests . Verification of the protein's dimeric structure can be performed using non-reducing versus reducing SDS-PAGE, with expected molecular weights of approximately 24 kDa and 12 kDa, respectively . Additionally, researchers should confirm the absence of aggregation, which can significantly impact bioactivity, through techniques such as size exclusion chromatography or dynamic light scattering.

What reconstitution and storage conditions maximize TGF-beta 2 stability and activity?

Proper reconstitution and storage of recombinant TGF-beta 2 is critical for maintaining its biological activity throughout experimental workflows. Lyophilized TGF-beta 2 is typically formulated in solutions such as 4mM HCl and should be reconstituted according to manufacturer specifications, with centrifugation of vials before opening to ensure all material is collected at the bottom of the tube . Researchers should avoid reconstituting to concentrations below 100 μg/ml as protein stability may be compromised at lower concentrations . When reconstituting the protein, gentle mixing rather than vortexing or vigorous pipetting is recommended to prevent denaturation of the protein structure . For short-term storage (1-2 weeks), reconstituted TGF-beta 2 can be kept at 4°C, while for long-term storage, aliquoting and storing at -20°C to -80°C is advised to avoid repeated freeze-thaw cycles that can degrade protein activity. Addition of carrier proteins such as bovine serum albumin (0.1-1%) can enhance stability during storage, particularly for dilute solutions used in cell culture experiments.

How should dosage and treatment duration be optimized for different experimental models?

Determining optimal dosage and treatment duration for TGF-beta 2 experiments requires careful consideration of the cellular context and specific research objectives. Based on published research, effective TGF-beta 2 concentrations for in vitro bioactivity typically range from 0.025-0.25 ng/mL for inhibition of cell proliferation to 1-10 ng/mL for induction of fibrotic responses . Treatment duration should be established through time-course experiments, as some TGF-beta 2 responses (like transcriptional changes) may occur within hours while others (such as matrix deposition or phenotypic alterations) may require days to become apparent. When designing experiments targeting fibrotic mechanisms, researchers have successfully used TGF-beta 2 at concentrations that induce expression of fibrotic genes in cholangiocytes and hepatic stellate cells over 24-72 hour periods . For in vivo applications, such as studying injury-induced death of mature motoneurons, treatment protocols must account for tissue distribution, half-life, and potential systemic effects of TGF-beta 2 . A dose-response curve should be established for each experimental system, as sensitivity to TGF-beta 2 varies considerably between different cell types and may be influenced by the expression levels of TGF-beta receptors.

What approaches effectively measure TGF-beta 2 activity in biological samples?

Multiple complementary approaches can be employed to measure TGF-beta 2 activity in biological samples, each with distinct advantages for specific research contexts. Quantitative detection of TGF-beta 2 protein levels is commonly achieved using enzyme-linked immunosorbent assays (ELISAs) with TGF-beta 2-specific antibodies, which can detect concentrations as low as 1-10 pg/mL in biological fluids or cell culture supernatants . For functional activity assessment, bioassays using HT-2 mouse T cell line proliferation inhibition provide a sensitive readout of TGF-beta 2 bioactivity with detectable effects at 0.025-0.25 ng/mL . Molecular signaling events can be monitored by measuring phosphorylation of downstream mediators like SMAD2/3 using western blot or phospho-specific flow cytometry. When analyzing tissue samples, researchers have successfully employed RNA sequencing to assess transcriptional responses to TGF-beta 2 or its silencing, as demonstrated in studies of liver fibrosis where differential expression analysis revealed TGF-beta 2-regulated genes involved in fibrosis and inflammation . Additionally, immunohistochemistry using isoform-specific antibodies can be used to localize TGF-beta 2 protein within tissue samples, though careful validation of antibody specificity is essential as commercial antibodies may exhibit cross-reactivity with other TGF-beta isoforms .

How can TGF-beta 2 silencing be implemented to study fibrogenic mechanisms?

TGF-beta 2 silencing represents a powerful approach for investigating fibrogenic mechanisms, particularly in cholestatic liver diseases where upregulated TGF-beta 2 contributes to pathogenesis. Antisense oligonucleotides (AONs) specifically targeting TGF-beta 2 have been successfully employed in mouse models of liver disease, resulting in significant reduction of collagen deposition, hydroxyproline content, and αSMA expression, all hallmarks of hepatic fibrosis . When designing TGF-beta 2 silencing experiments, researchers should consider cell type-specific targeting, as AONs primarily affect liver sinusoidal endothelial cells, activated fibroblasts, and macrophages rather than hepatocytes . Validation of knockdown efficiency should be performed at both mRNA level (using qRT-PCR) and protein level (using western blot or ELISA) to confirm the degree of TGF-beta 2 suppression achieved. Beyond fibrosis markers, researchers should assess broader consequences of TGF-beta 2 silencing, including effects on inflammatory gene expression (e.g., Ccl3, Ccl4, Ccl5) and tissue infiltration by immune cells, as TGF-beta 2 knockdown in MDR2-KO mice decreased CD45-positive inflammatory cell infiltration while increasing F4/80-positive cells including eosinophils .

What methodologies are effective for studying TGF-beta 2 in cardiovascular disease research?

Investigation of TGF-beta 2 in cardiovascular disease contexts requires specialized methodologies that capture its role in atherosclerotic plaque stability and progression. Quantification of TGF-beta isoforms in human carotid plaques has been effectively performed using specific immunoassays, revealing TGF-beta 2 as the most abundant isoform with significant associations to plaque stability features . Transcriptomic analysis through RNA sequencing of plaque tissue provides valuable insights into TGF-beta 2 expression patterns, with researchers successfully employing Ribo-Zero™ Magnetic Kit for ribosomal RNA clearance followed by strand-specific RNAseq library preparation . When analyzing relationships between TGF-beta 2 and clinical outcomes, survival analyses using Kaplan-Meier curves with Log-rank test and Cox proportional hazard regression models can identify associations between TGF-beta 2 levels and cardiovascular event risk, as demonstrated in studies where patients with plaques containing high TGF-beta 2 levels showed lower risk of future cardiovascular events . For in vitro mechanistic studies, THP-1 and RAW264.7 macrophage cell lines treated with recombinant TGF-beta 2 have been used to assess effects on inflammation markers and protease activity relevant to atherosclerotic processes .

How can researchers differentiate between TGF-beta 2-specific effects and those common to all TGF-beta isoforms?

Distinguishing TGF-beta 2-specific effects from those shared across all TGF-beta isoforms requires careful experimental design incorporating multiple complementary approaches. Isoform-specific knockdown or knockout models provide the most definitive evidence of TGF-beta 2-specific functions, as demonstrated in mouse studies where targeted deletion of TGF-beta 2 revealed non-redundant roles in cardiac, lung, craniofacial, and other organ development that were not compensated by other isoforms . When working with recombinant proteins, parallel experiments with equivalent concentrations of TGF-beta 1, TGF-beta 2, and TGF-beta 3 can identify differential responses, using readouts such as transcriptional profiling, phospho-proteomics, or functional assays. Researchers should be aware that commercial anti-TGF-beta antibodies often exhibit cross-reactivity between isoforms, necessitating careful validation of antibody specificity before attribution of effects to specific isoforms . Expression analysis in disease contexts can reveal differential regulation patterns of TGF-beta isoforms, as observed in liver disease studies where TGF-beta 1 and TGF-beta 2 showed markedly different expression patterns, suggesting distinct roles in pathogenesis .

How can researchers address variable responses to TGF-beta 2 in cell culture systems?

Variable cellular responses to TGF-beta 2 in experimental systems often stem from several key factors that researchers should systematically address. Cell density significantly impacts TGF-beta 2 responsiveness, with confluent cultures typically showing diminished sensitivity compared to sub-confluent cultures; therefore, standardizing seeding density and treating cells at consistent confluence levels across experiments is crucial . The presence of endogenous TGF-beta in serum can mask exogenous TGF-beta 2 effects, making serum reduction or the use of TGF-beta-depleted serum advisable during treatment periods. Receptor expression varies considerably between cell types and culture conditions, so researchers should verify TGF-beta receptor levels (particularly TβRII and TβRI/ALK5) in their experimental system through flow cytometry or western blotting before interpreting TGF-beta 2 response data. The activation state of latent TGF-beta 2 is critical, as the recombinant protein often exists in a latent complex with LAP, requiring activation for signaling; researchers can ensure activation through acidification (pH 2-3 for 30 minutes followed by neutralization) or using proteases like plasmin prior to cell treatment . Additionally, cross-talk with other signaling pathways (such as Notch, Wnt/β-catenin, or inflammatory cytokines) can profoundly modify TGF-beta 2 responses, necessitating careful control of the cellular microenvironment during experiments.

What challenges occur when measuring TGF-beta 2 in complex biological samples?

Accurate measurement of TGF-beta 2 in complex biological samples presents several technical challenges requiring specific methodological solutions. The presence of TGF-beta binding proteins in biological fluids can mask epitopes or sequester TGF-beta 2, leading to underestimation of levels; sample acidification (pH 2-3) followed by neutralization before assay can release TGF-beta 2 from these complexes, revealing the total TGF-beta 2 pool . Matrix effects from plasma, serum, or tissue homogenates often interfere with immunoassays, necessitating spike-and-recovery validation experiments with known quantities of recombinant TGF-beta 2 to verify assay accuracy in each sample type . Cross-reactivity between TGF-beta isoforms can confound results when using non-specific antibodies, making it essential to select isoform-specific detection methods and validate antibody specificity through western blotting with recombinant TGF-beta 1, 2, and 3 proteins . Low abundance of TGF-beta 2 in some biological samples can challenge detection limits of standard assays, requiring sample concentration techniques or highly sensitive detection methods such as digital ELISA platforms. For tissue samples, proper extraction procedures are critical as suboptimal methods may fail to liberate TGF-beta 2 from extracellular matrix associations, as demonstrated in studies of human carotid plaques where specialized extraction protocols were needed to accurately quantify TGF-beta isoforms .

What strategies help distinguish between active and latent forms of TGF-beta 2?

Distinguishing between active and latent forms of TGF-beta 2 is crucial for accurate interpretation of experimental results and requires specific methodological approaches. Bioassays using TGF-beta-responsive cell lines provide a functional readout of only the active TGF-beta 2 fraction, as the latent form cannot engage receptors without prior activation; comparing results from samples with and without acid activation (pH 2-3 for 30 minutes followed by neutralization) allows quantification of both total and active TGF-beta 2 pools . Specialized immunoassays with antibodies specifically recognizing either the active form or the LAP portion of TGF-beta 2 can directly quantify each pool, though careful validation of antibody specificity is essential . Native gel electrophoresis under non-reducing conditions can separate the ~24 kDa active dimeric TGF-beta 2 from the higher molecular weight latent complexes, followed by western blotting with specific antibodies for visualization . For research focused on activation mechanisms, experimental designs should incorporate assays for activators such as integrins, thrombospondin-1, or proteases including plasmin and matrix metalloproteinases, which are physiologically relevant converters of latent to active TGF-beta 2 . When working with recombinant TGF-beta 2, researchers should verify the form provided by manufacturers, as some products are pre-activated while others require activation before use in cellular assays .

How is TGF-beta 2 being targeted in experimental disease therapies?

TGF-beta 2 targeted therapies represent a promising approach for treating fibrotic and inflammatory conditions, with several strategic approaches under investigation. Antisense oligonucleotides (AONs) specifically targeting TGF-beta 2 have demonstrated significant therapeutic potential in cholestatic liver disease models, where treatment led to reduced collagen deposition, decreased hydroxyproline content, and diminished αSMA expression without adverse effects on healthy liver tissue . When designing TGF-beta 2-targeting therapeutic strategies, researchers must consider tissue-specific effects, as TGF-beta 2 silencing in MDR2-KO mice showed regulatory effects on inflammatory genes (Ccl3, Ccl4, Ccl5) and altered tissue infiltration by immune cells, suggesting complex immunomodulatory consequences beyond direct antifibrotic effects . The correlation between TGF-beta 2 expression and disease progression markers, such as the positive correlation between TGFB2 and CD45 expression in primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients, provides rationale for monitoring these parameters as surrogate endpoints in therapeutic trials . In cardiovascular disease contexts, TGF-beta 2's association with plaque stability suggests caution in therapeutic targeting, as studies found patients with higher plaque TGF-beta 2 levels had lower risk of future cardiovascular events, indicating potential benefits of maintaining or enhancing TGF-beta 2 activity in atherosclerotic disease .

What emerging approaches are advancing the study of TGF-beta 2 signaling specificity?

Cutting-edge technologies are enhancing our understanding of TGF-beta 2 signaling specificity across different biological contexts. Single-cell RNA sequencing now enables researchers to delineate cell type-specific responses to TGF-beta 2 within heterogeneous tissues, revealing previously unappreciated variability in signaling outcomes between neighboring cells exposed to identical TGF-beta 2 concentrations . CRISPR/Cas9-based gene editing facilitates precise modification of TGF-beta 2 ligand, receptor components, or downstream effectors, allowing systematic dissection of signaling pathway elements that determine isoform-specific versus shared responses. Phospho-proteomics approaches can comprehensively map signaling networks activated by TGF-beta 2 compared to other isoforms, potentially identifying divergent pathways that explain non-redundant functions. Advanced imaging techniques including proximity ligation assays and fluorescence resonance energy transfer (FRET) enable visualization of TGF-beta 2-specific protein-protein interactions in live cells, providing spatial and temporal resolution of signaling events. Computational modeling integrating transcriptomic, proteomic, and functional data shows promise for predicting context-dependent outcomes of TGF-beta 2 signaling, as demonstrated in studies where Orthogonal Projections to Latent Structures Discriminant Analysis identified TGF-beta 2 as a key determinant separating symptomatic from asymptomatic atherosclerotic plaques .

How can TGF-beta 2 serve as a biomarker for disease progression or therapeutic response?

TGF-beta 2's potential as a biomarker spans multiple disease contexts, with particular promise in fibrotic conditions and cardiovascular pathologies. In atherosclerotic disease, TGF-beta 2 levels in carotid plaques have shown significant prognostic value, with higher levels correlating with plaque stability features and lower risk of future cardiovascular events as demonstrated through Kaplan-Meier survival analyses and Cox proportional hazard regression models . For monitoring purposes, both tissue and circulating TGF-beta 2 levels may provide valuable information, though measurement standardization is essential with techniques such as immunoassays validated for specific sample types through spike-and-recovery experiments . When evaluating TGF-beta 2 as a response biomarker in therapeutic trials, researchers should consider measuring both the protein itself and downstream effects such as SMAD phosphorylation or target gene expression, providing a more complete picture of pathway modulation. The ratio between active and total TGF-beta 2 may offer more informative biomarker value than absolute levels alone, particularly in conditions where dysregulated activation rather than expression drives pathology . Integrated biomarker panels combining TGF-beta 2 with related molecules such as matrix metalloproteinases or inflammatory cytokines may provide enhanced predictive power, as suggested by correlation studies showing TGF-beta 2's inverse relationship with matrix-degrading matrix metalloproteinase-9 and inflammatory markers in plaque tissue .