VEGF Mouse (121 a.a.), Yeast

What is VEGF Mouse (121 a.a.) and how does it differ structurally from other VEGF isoforms?

VEGF Mouse (121 a.a.) is a specific isoform of Vascular Endothelial Growth Factor A (VEGF-A) that consists of 121 amino acids. It is a disulfide-linked homodimer with approximately 20.7kDa molecular mass per polypeptide chain . This isoform is also known by several alternative names including Vascular endothelial growth factor A, VEGF-A, Vascular permeability factor (VPF), and MGC70609 .

The amino acid sequence of Mouse VEGF (121 a.a.) is: MAPTTEGEQK SHEVIKFMDV YQRSYCRPIE TLVDIFQEYP DEIEYIFKPS CVPLMRCAGC CNDEALECVP TSESNITMQI MRIKPHQSQH IGEMSFLQHS RCECRPKKDR TKPEKCDKPR R .

Unlike the larger VEGF isoforms (such as VEGF165 and VEGF189), VEGF121 lacks the heparin-binding domain, which affects its interaction with extracellular matrix (ECM) components. This structural difference causes VEGF121 to be more freely diffusible compared to larger isoforms that remain anchored to the ECM .

What are the optimal storage and reconstitution conditions for yeast-produced VEGF Mouse (121 a.a.)?

For optimal stability and bioactivity retention, the following storage and reconstitution protocols should be observed:

Storage conditions:

• Lyophilized VEGF (121 a.a.) is stable at room temperature for up to 3 weeks

• For long-term storage, keep desiccated below -18°C

• Avoid repeated freeze-thaw cycles

Reconstitution protocol:

• Reconstitute the lyophilized protein in sterile 18MΩ-cm H₂O at a concentration not less than 100μg/ml

• The reconstituted solution can be further diluted to other aqueous solutions as needed

• After reconstitution, store at 4°C for use within 2-7 days

• For future use beyond 7 days, aliquot and store below -18°C

Following these guidelines ensures maximum retention of biological activity and prevents protein degradation that could compromise experimental results.

How is VEGF Mouse (121 a.a.) efficiently produced in yeast expression systems?

The production of VEGF Mouse (121 a.a.) in yeast involves several critical steps:

• Vector construction: The coding sequence for VEGF121 is typically cloned into a suitable yeast expression vector. For example, as seen in similar VEGF isoform studies, pPICZalphaC vector can be used for production in Pichia pastoris, where the alpha-factor signal peptide facilitates extracellular expression .

• Transformation and clone selection: The expression vector is introduced into the yeast host (typically Saccharomyces cerevisiae), and transformants are selected based on appropriate markers .

• Growth conditions: Selected yeast clones are grown in suitable media such as BMGY (Buffered Glycerol-complex Medium) at approximately 29°C with constant shaking for optimal cell density .

• Protein induction: Protein expression is induced by transferring cells to an induction medium such as BMMY (Buffered Methanol-complex Medium), where methanol replaces glycerol as the carbon source .

• Harvest and purification: After approximately 24 hours of induction, the culture supernatant containing the secreted protein is collected and purified using chromatographic techniques, most commonly nickel-affinity chromatography for His-tagged proteins .

The resulting product is a sterile filtered white lyophilized powder of high purity (>95%) as determined by RP-HPLC and SDS-PAGE analysis .

What quality control parameters should be assessed for recombinant VEGF Mouse (121 a.a.)?

To ensure experimental reproducibility and reliability, the following quality control parameters should be evaluated:

• Purity assessment:

  • Analysis by RP-HPLC (Reverse Phase High-Performance Liquid Chromatography)

  • Analysis by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)

  • Aim for greater than 95.0% purity

• Biological activity:

  • In vitro kinase assays to examine phosphorylation of VEGFR2

  • Endothelial cell proliferation assays

  • Migration assays to confirm functional activity

• Identity confirmation:

  • Mass spectrometry analysis

  • Western blot with specific antibodies

  • N-terminal sequencing to confirm protein identity

• Endotoxin testing:

  • Limulus Amebocyte Lysate (LAL) assay to ensure minimal endotoxin contamination

These rigorous quality control measures help ensure that experimental outcomes are not influenced by contaminants or variations in protein quality.

What are the key biological functions of VEGF Mouse (121 a.a.) in angiogenesis research?

VEGF Mouse (121 a.a.) exhibits several crucial biological functions that make it valuable for angiogenesis research:

• Vascular permeability: VEGF121 functions as a vasodilator and increases microvascular permeability, which is why it was originally referred to as vascular permeability factor (VPF) .

• Endothelial cell effects:

  • Stimulates endothelial cell mitogenesis

  • Promotes endothelial cell migration

  • Inhibits endothelial cell apoptosis

  • Induces endothelial cell growth

• Vessel formation: VEGF121 plays crucial roles in both angiogenesis (formation of new blood vessels from existing ones) and vasculogenesis (de novo formation of the embryonic circulatory system) .

• Non-endothelial effects: While VEGF activity has been predominantly studied in vascular endothelium, it also affects other cell types, including:

  • Monocyte/macrophage migration

  • Neurons

  • Cancer cells

  • Kidney epithelial cells

Understanding these functions is essential for designing experiments that accurately investigate angiogenic processes and developing therapeutic strategies targeting the VEGF pathway.

How can VEGF Mouse (121 a.a.) be used in tumor angiogenesis models?

VEGF Mouse (121 a.a.) has proven valuable in developing tumor angiogenesis models, particularly through the following approaches:

• Endothelial cell transformation: Overexpression of VEGF121 in immortalized endothelial cells, such as the MS1 cell line derived from murine cells using temperature-sensitive SV40 large T antigen, results in the development of slowly growing angiosarcomas . This model provides insights into:

  • VEGF-driven vascular tumor formation

  • Well-differentiated angiosarcoma development

  • The role of VEGF in endothelial cell malignant transformation

• Receptor regulation: MS1 VEGF cells developed from this approach demonstrate up-regulation of VEGF receptors VEGFR-2 (Flk-1/Kdr) and VEGFR-1 (Flt-1), providing a system to study receptor dynamics in tumor development .

• Comparative oncogenesis: This model allows comparison with other angiosarcoma models, such as those developed through the introduction of activated H-Ras, enabling investigation of different pathways to endothelial malignancy .

The biological activity of VEGF produced in these models can be confirmed through in vitro kinase assays examining phosphorylation of VEGFR-2 on intact cells in response to exogenously added VEGF .

How does matrix-bound VEGF differ from soluble VEGF in receptor activation patterns?

The interaction between VEGF and the extracellular matrix (ECM) significantly influences its signaling properties. Though VEGF121 lacks the primary heparin-binding domain present in larger isoforms, understanding these differences remains relevant for comparative research:

• Phosphorylation duration: Matrix-bound VEGF elicits more prolonged VEGFR2 activation compared to soluble VEGF, with extended phosphorylation often observed in matrix-bound contexts .

• Tyrosine phosphorylation patterns: Different tyrosine residues on VEGFR2 show distinct phosphorylation patterns when activated by matrix-bound versus soluble VEGF:

  • Y951 shows initial preferential phosphorylation by matrix-bound VEGF but is quickly dephosphorylated (by 15 minutes)

  • Y1214 demonstrates substantial retention of activation exclusively by matrix-bound VEGF, with gradual return to baseline only after 30 minutes

  • This prolonged Y1214 phosphorylation appears responsible for the extended activation observed with matrix-bound VEGF

• Matrix protein influence: Different collagen types (I, IV, V, and VIII) can mediate VEGF immobilization, though with varying levels of phosphorylation retention at Y1214 .

These differential activation patterns suggest that the presentation context of VEGF (soluble vs. matrix-bound) may be as important as the presence of the growth factor itself in determining biological outcomes in research models.

What receptor-level mechanisms explain VEGF121's biological activity?

The biological activity of VEGF121 involves several receptor-level mechanisms that can be demonstrated experimentally:

• Receptor autophosphorylation: VEGF121 binding to VEGFR2 initiates receptor autophosphorylation, which can be assessed through in vitro kinase assays on intact cells .

• Chronic receptor stimulation effects: Cells continuously exposed to VEGF121 (such as those engineered to overexpress it) demonstrate:

  • Decreased response to exogenous VEGF due to receptor blockade by endogenously produced VEGF

  • Increased receptor turnover resulting from chronic stimulation

  • Intracellular binding of endogenously produced VEGF121 to VEGFR2 within cellular compartments like the endoplasmic reticulum or Golgi apparatus

• Receptor degradation pathways: Chronic activation of VEGFR2 by VEGF121 leads to receptor degradation through:

  • Lysosomal degradation pathways

  • Ubiquitination and proteasome-mediated degradation

These mechanisms explain why cells chronically exposed to VEGF121 might not show increased levels of autophosphorylated receptors despite ongoing stimulation.

What are the optimal methodologies for assessing VEGF Mouse (121 a.a.) biological activity?

Several complementary approaches can be used to assess the biological activity of VEGF Mouse (121 a.a.):

• Receptor phosphorylation assays:

  • In vitro kinase assays examining phosphorylation of VEGFR2 on intact cells

  • Western blotting with phospho-specific antibodies (4G10 pan-phosphotyrosine antibody and antibodies to specific VEGFR2 phosphotyrosine residues like Y951 and Y1214)

• Receptor mutation studies:

  • Using cells transfected with VEGFR2 mutants (Y1214F, Y951F, Y1130F)

  • Comparing phosphorylation patterns between wild-type and mutant receptors to identify key signaling residues

• Metabolic labeling:

  • Metabolic labeling of cells with radioisotopes to track receptor expression and turnover

  • Immunoprecipitation of labeled receptors to assess dynamics

• Functional assays:

  • Endothelial cell proliferation assays

  • Migration assays

  • Tube formation assays in Matrigel

  • In vivo angiogenesis assays such as Matrigel plug assays or chorioallantoic membrane assays

These methodologies provide complementary information about different aspects of VEGF biological activity, from immediate receptor activation to downstream functional consequences.

How should researchers design experiments to investigate VEGF121 versus other isoforms?

When designing comparative experiments between VEGF121 and other isoforms (like VEGF165 or VEGF189), several methodological considerations are important:

• Expression system consistency:

  • Use the same expression system (e.g., yeast) for all isoforms being compared

  • Apply identical purification protocols to eliminate system-specific variations

• Concentration normalization:

  • Normalize protein concentrations accurately

  • Account for differences in molecular weight when comparing molar concentrations

  • Validate through ELISA or other quantitative methods

• Matrix interaction controls:

  • Design experiments that account for differential matrix binding properties

  • Include both soluble and matrix-bound presentation formats

  • Use matrix types relevant to the research question (e.g., different collagen types)

• Receptor expression verification:

  • Confirm expression levels of relevant receptors (VEGFR1, VEGFR2) in experimental cells

  • Consider co-receptors like neuropilins that may influence isoform-specific signaling

• Readout diversity:

  • Employ multiple complementary readouts (phosphorylation patterns, calcium signaling, proliferation, migration)

  • Include time course analyses to capture transient versus sustained effects

Following these guidelines helps ensure that observed differences between VEGF isoforms reflect true biological variation rather than experimental artifacts.

What are common issues in VEGF Mouse (121 a.a.) experiments and how can they be addressed?

Researchers working with VEGF Mouse (121 a.a.) may encounter several challenges that can be addressed through specific troubleshooting approaches:

• Loss of biological activity:

  • Cause: Protein degradation during storage or handling

  • Solution: Strictly adhere to recommended storage conditions; avoid repeated freeze-thaw cycles; add carrier proteins like BSA for dilute solutions

• Inconsistent phosphorylation results:

  • Cause: Variations in receptor expression or pre-existing activation

  • Solution: Serum-starve cells prior to experiments; confirm receptor expression levels; use positive controls with commercial VEGF

• Differential responses between cell types:

  • Cause: Varying expression levels of VEGF receptors and co-receptors

  • Solution: Characterize receptor expression profiles before experiments; consider transfection to normalize receptor levels

• Matrix interaction variability:

  • Cause: Batch-to-batch variation in matrix proteins

  • Solution: Use defined synthetic matrices when possible; characterize new batches of natural matrices

• Endotoxin contamination:

  • Cause: Introduction during purification or handling

  • Solution: Use endotoxin-free reagents; test preparations with LAL assay; include polymyxin B controls in critical experiments

Addressing these issues proactively ensures more reliable and reproducible experimental outcomes.

How can VEGF Mouse (121 a.a.) be used in advanced angiogenesis research applications?

VEGF Mouse (121 a.a.) can be utilized in several advanced research applications:

• Transgenic and knock-in models:

  • Developing models with cell-type specific or inducible VEGF121 expression

  • Creating receptor mutant models to study isoform-specific signaling

  • Using these models to investigate developmental angiogenesis or pathological conditions

• 3D tissue engineering:

  • Incorporation into biocompatible scaffolds at controlled concentrations

  • Development of gradient systems to study directional vessel formation

  • Creating organoid systems with regulated VEGF presentation

• Disease modeling:

  • Investigations of VEGF121's role in diabetic retinopathy

  • Studies on POEMS syndrome (Crow-Fukase syndrome), which is linked to elevated VEGF levels

  • Cancer angiogenesis models comparing efficacy of anti-VEGF therapeutics against different isoforms

• Combination therapies:

  • Studying synergistic effects with other growth factors (FGF, PDGF)

  • Investigating interactions with ECM-modifying enzymes

  • Exploring combinations with emerging immunotherapeutic approaches

These advanced applications extend beyond basic research and have translational potential for addressing pathological angiogenesis in clinical settings.