VEGF (121a.a.) Human, HEK

What is VEGF (121a.a.) Human, HEK and how does it differ from other VEGF isoforms?

VEGF (121a.a.) Human, HEK refers to the 121 amino acid isoform of Vascular Endothelial Growth Factor, produced in Human Embryonic Kidney cells. This isoform is one of several splice variants of the VEGF-A gene, distinguished by lacking heparin-binding domains found in larger isoforms. The HEK expression system provides human-like post-translational modifications compared to bacterial expression systems. VEGF (121a.a.) primarily functions as a potent stimulator of endothelial cell proliferation and migration, with critical roles in angiogenesis and vascular permeability. Its absence of heparin-binding domains results in higher diffusibility compared to VEGF165 or VEGF189 isoforms, making it particularly useful for research requiring soluble growth factors that can freely diffuse through tissues .

What structural and stability characteristics should researchers consider when working with VEGF (121a.a.) Human, HEK?

When working with VEGF (121a.a.) Human, HEK, researchers should consider several key stability parameters:

• Storage conditions: Lyophilized VEGF remains stable at room temperature for approximately 3 weeks but should be stored desiccated below -18°C for long-term preservation .

• Reconstitution guidelines: It is recommended to reconstitute lyophilized VEGF in sterile PBS containing 0.1% endotoxin-free recombinant human serum albumin (HSA) .

• Working solution stability: Upon reconstitution, VEGF should be stored at 4°C if used within 2-7 days, or below -18°C for future use .

• Carrier protein addition: For long-term storage, adding a carrier protein (0.1% HSA or BSA) is recommended to maintain stability .

• Freeze-thaw sensitivity: Repeated freeze-thaw cycles should be avoided as they can significantly impact biological activity .

The physical appearance of properly stored VEGF (121a.a.) Human, HEK is typically a sterile filtered white lyophilized powder with greater than 95% purity as observed by SDS-PAGE analysis .

How can molecular chaperones enhance the folding and solubility of recombinant VEGF (121a.a.)?

Molecular chaperones play a crucial role in enhancing the folding and solubility of recombinant VEGF (121a.a.). When expressing VEGF in bacterial systems like Escherichia coli, co-expression with molecular chaperones can significantly improve proper protein folding. Research has demonstrated that co-expression with GroES/EL molecular chaperones (encoded by plasmid pGro7) produced VEGF variants with 3.9-8.7 times greater mitogenic activity compared to commercially available variants .

The methodological approach involves:

• Constructing a fusion protein (e.g., with thioredoxin) to improve solubility

• Using specialized E. coli strains like Origami B(DE3) that enhance disulfide bond formation

• Co-transforming cells with a chaperone-encoding plasmid like pGro7

• Optimizing expression conditions:

  • Lowering temperature to 25°C

  • Using low IPTG concentration (0.02 mM)

  • Inducing chaperone expression with arabinose (1.7 g/L)

This strategy effectively addresses the challenging aspects of expressing disulfide-rich proteins like VEGF in bacterial systems while maintaining their biological activity .

What expression and purification strategies yield the highest quality VEGF (121a.a.)?

High-quality VEGF (121a.a.) production requires careful consideration of expression systems and purification strategies. While HEK-derived VEGF provides native-like post-translational modifications, optimized bacterial expression systems can offer cost-effective alternatives with enhanced activity.

For optimized bacterial expression:

• Vector selection: pET-32a(+) vector containing thioredoxin fusion partner improves solubility and folding

• Host strain: E. coli Origami B(DE3) enhances disulfide bond formation

• Chaperone co-expression: GroES/EL chaperones significantly improve folding

• Culture conditions: Growth at 37°C until OD600 reaches 0.5, followed by temperature reduction to 25°C

• Induction parameters: Low IPTG concentration (0.02 mM) and arabinose (1.7 g/L) for chaperone induction

Purification typically involves:

• Affinity chromatography of the soluble fraction

• Thrombin cleavage to remove the fusion partner

• Second round of affinity chromatography to isolate the target protein

This approach has yielded purified α2-PI 1-8-VEGF 121 at approximately 1.4 mg per liter of cell culture with significantly enhanced mitogenic activity compared to commercially available variants .

What are the most reliable methods to quantify the biological activity of VEGF (121a.a.)?

Quantifying the biological activity of VEGF (121a.a.) requires methodologies that reflect its primary functions. The gold standard approach involves measuring its effect on endothelial cell proliferation, particularly using Human Umbilical Vein Endothelial Cells (HUVEC).

Two complementary methods are recommended:

• Real-time cell analysis using electrical impedance measurement systems (e.g., xCELLigence)

  • Advantages: Provides continuous, label-free monitoring of cell proliferation

  • Protocol: Seed 3,500 HUVEC cells per well in appropriate medium with VEGF concentrations ranging from 20-100 ng/mL

  • Monitoring: Cell growth can be tracked every 15 minutes for up to 7 days

• Metabolic activity assays (e.g., resazurin-based)

  • Advantages: Endpoint measurement of cellular metabolic activity

  • Applications: Useful for high-throughput screening of multiple conditions

For rigorous quantification, researchers should:

• Establish dose-response curves using multiple VEGF concentrations (20, 50, and 100 ng/mL)

• Determine the effective dose producing 50% maximal response (ED50)

• Include appropriate positive controls (commercial VEGF standards) and negative controls (basal media without growth factors)

• Control for potential contaminants like thrombin in parallel experiments

The specific activity of high-quality VEGF (121a.a.) Human, HEK is typically determined by dose-dependent stimulation of HUVEC proliferation, with an ED50 of approximately 3 ng/mL .

How does the mitogenic activity of different VEGF (121a.a.) preparations compare in endothelial cell assays?

Different VEGF (121a.a.) preparations can exhibit significant variations in mitogenic activity. Research has shown that optimized expression systems incorporating molecular chaperones can produce VEGF variants with substantially higher activity than commercial preparations.

Comparative activity data shows:

This activity difference is most pronounced at a concentration of 50 ng/mL of VEGF, though it remains observable across the typical working range of 20-100 ng/mL .

When designing experiments, researchers should consider these activity differences and standardize their VEGF concentrations based on biological activity rather than protein mass alone. This is especially important when comparing results across different studies that may have used different VEGF preparations .

How can VEGF (121a.a.) be modified for incorporation into biomaterials for tissue engineering?

Modification of VEGF (121a.a.) for incorporation into biomaterials represents an advanced application with significant implications for tissue engineering and regenerative medicine. One effective approach involves adding specific peptide sequences to enable covalent anchoring within biomaterials.

The α2-PI 1-8-VEGF 121 variant provides an excellent example of this strategy:

• Modification approach: Addition of a substrate sequence (NQEQVSPL) for factor XIIIa at the aminoterminus of VEGF 121

• Functional advantage: Enables covalent incorporation of VEGF into fibrin networks through enzymatic cross-linking

• Applications: VEGF-loaded fibrin matrices have demonstrated increased growth activity of vascular endothelial cells

• Clinical potential: These matrices can be used for coating vascular prostheses, stents, or heart valve replacements to accelerate endothelialization

Production of such modified variants requires careful design:

• Gene synthesis with optimized codon usage for the expression system

• Cloning into appropriate vectors (e.g., pET-32a(+) with thioredoxin fusion)

• Expression in systems that ensure proper folding (E. coli with chaperones or mammalian cells)

• Verification of maintained biological activity after modification

Importantly, the modified α2-PI 1-8-VEGF 121 variant maintained high mitogenic activity without eliciting significant inflammatory activation of endothelial or monocyte-like cells, making it particularly valuable for tissue engineering applications .

What experimental considerations are important when studying VEGF (121a.a.) effects on inflammation and immune cell activation?

When studying VEGF (121a.a.) interactions with the immune system, researchers must consider several experimental design factors to obtain reliable and physiologically relevant results.

Key experimental considerations include:

• Cell type selection:

  • Endothelial cells (e.g., HUVEC) for vascular responses

  • Monocyte-like cells (e.g., THP-1) for immune activation assessment

  • Primary human cells versus established cell lines (trade-offs between consistency and physiological relevance)

• Inflammatory marker assessment:

  • Cytokine profiling should include multiple markers (IL-1α, IL-1β, TNF-α, IL-8, MCP-1, GM-CSF)

  • Time-course studies are critical as responses may be transient

  • Appropriate positive controls (e.g., lipopolysaccharide) should be included

• Concentration considerations:

  • Test multiple VEGF concentrations (typically 20-50 ng/mL for in vitro studies)

  • Consider dose-response relationships

• Potential confounding factors:

  • Contaminants in VEGF preparations (endotoxin, thrombin)

  • Serum components in culture media

  • Cell passage number and culture conditions

Research has shown that highly purified α2-PI 1-8-VEGF 121 did not elicit production of IL-1α, IL-1β, and GM-CSF in THP-1 cells at detectable levels, while it did stimulate TNF-α, MCP-1, and IL-8 production in a concentration-dependent but transient manner, primarily observed during the first 3 days of culture .

How do contaminating factors in VEGF (121a.a.) preparations affect experimental results?

Contaminating factors in VEGF preparations can significantly impact experimental outcomes and lead to misinterpretation of results. Researchers should be aware of these potential confounders:

• Thrombin contamination:

  • Origin: Often used for cleavage of fusion proteins during purification

  • Impact: Can stimulate endothelial cell proliferation independently of VEGF

  • Control strategy: Include parallel experiments with corresponding thrombin concentrations

  • Assessment: When testing VEGF at 20-100 ng/mL, evaluate thrombin effects at concentrations ranging from 0.01-1.0 NIH U/mL

• Endotoxin contamination:

  • Origin: Bacterial expression systems, particularly E. coli

  • Impact: Potent inducer of inflammatory responses (IL-1α, IL-1β, TNF-α, IL-8)

  • Control strategy: Rigorous endotoxin testing and removal during purification

  • Assessment: Include lipopolysaccharide controls in immune activation studies

• Fusion protein remnants:

  • Origin: Incomplete cleavage during purification

  • Impact: May alter binding properties or introduce unintended biological activities

  • Control strategy: Thorough characterization by SDS-PAGE and Western blotting

To address these challenges, researchers should implement:

• Multiple purification steps (e.g., sequential chromatography)

• Endotoxin testing of final preparations

• Validation of biological activity with appropriate positive and negative controls

• Quality control testing for batch-to-batch consistency

What factors can lead to batch-to-batch variation in VEGF (121a.a.) activity, and how can these be minimized?

Batch-to-batch variation in VEGF (121a.a.) activity presents a significant challenge for research reproducibility. Understanding and controlling these factors is essential for consistent experimental results.

Key sources of variation include:

• Expression system variables:

  • Cell culture conditions (temperature, medium composition, induction timing)

  • Expression strain characteristics and plasmid stability

  • Chaperone co-expression efficiency

  • Control strategy: Standardize culture conditions and monitor growth parameters (OD600)

• Purification process variations:

  • Chromatography conditions and column performance

  • Buffer composition and pH

  • Cleavage efficiency for fusion proteins

  • Control strategy: Establish detailed standard operating procedures and quality control checkpoints

• Protein stability factors:

  • Storage conditions (temperature, buffer composition)

  • Freeze-thaw cycles

  • Protein concentration effects

  • Control strategy: Implement consistent storage protocols with aliquoting to avoid freeze-thaw cycles

• Activity assessment standardization:

  • Cell passage number and source

  • Assay conditions and reagents

  • Control strategy: Include internal standards in each activity assay

To minimize batch-to-batch variation, researchers should:

• Implement comprehensive quality control testing for each batch

• Measure protein concentration by multiple methods (UV spectroscopy, Bradford assay, ELISA)

• Assess purity by SDS-PAGE (should exceed 95%)

• Determine specific activity through standardized bioassays

• Establish acceptance criteria for each quality attribute

• Create reference standards for comparative analysis