PLGF3 Human, sf9

What is PlGF-3 and how does it differ from other PlGF isoforms?

PlGF-3 is the third isoform of human Placental Growth Factor, a member of the vascular endothelial growth factor (VEGF) family that promotes endothelial cell growth and angiogenesis. The human PlGF gene is located on chromosome 14q24 and spans an 800-kb-long DNA segment comprising seven exons .

Unlike PlGF-2, which contains a heparin-binding domain, PlGF-3 lacks this domain while maintaining its ability to signal through the VEGFR-1 (FLT1) receptor . PlGF-3 is a glycosylated homodimer containing two chains of 203 amino acids (Leu19-Arg221) with a molecular mass of approximately 58 kDa when expressed in Sf9 cells . In contrast, PlGF-1 produced in insect cells is a homodimer containing 2 x 131 amino acids with a total molecular mass of approximately 34 kDa .

The key structural and functional differences between PlGF isoforms affect their secretion patterns, binding affinities, and biological activities, making each isoform uniquely suited for specific experimental applications.

What are the molecular characteristics of recombinant human PlGF-3?

Recombinant human PlGF-3 expressed in Sf9 insect cells has the following molecular characteristics:

The recombinant protein lacks a heparin-binding domain that is present in PlGF-2, which influences its interaction with cell surface proteoglycans and extracellular matrix components . This characteristic makes PlGF-3 particularly useful for studying the direct receptor-mediated signaling pathways without the confounding effects of heparin binding.

How is PlGF-3 involved in physiological and pathological processes?

PlGF-3, like other PlGF isoforms, appears to be dispensable for normal development and health but plays diverse roles in pathological conditions. Research indicates PlGF is involved in:

• Angiogenesis and Endothelial Cell Function: PlGF-3 stimulates proliferation and migration of endothelial cells, contributing to blood vessel formation .

• Placental Development: Higher amounts of PlGF-3 have been detected in placental tissue compared to serum, suggesting a specialized role in placentation .

• Retinal Function: Studies showed that PlGF gene knockdown in human retinal pigment epithelial (RPE) cells significantly reduced vascular tube formation in human umbilical vein endothelial cells (HUVECs), indicating PlGF's role as a key modulator of angiogenic potential in the human retina .

• Pathological Conditions: While PlGF-3's specific role in disease states is still being investigated, other PlGF isoforms have been implicated in pre-eclampsia, cancer progression, inflammation, and tissue ischemia .

Research indicates that PlGF-3 protein levels in maternal serum have been investigated for potential associations with pre-eclampsia (PE) and small for gestational age (SGA), though studies found that most serum samples had PlGF-3 levels below the detection limit of 1.6 pg/mL, with no predictive association for PE or SGA .

What is the optimal protocol for expressing PlGF-3 in Sf9 insect cells?

The optimal protocol for expressing PlGF-3 in Sf9 insect cells involves several critical steps:

• Cell Culture Preparation:

  • Grow Sf9 cells in suspension at 27°C in appropriate medium (e.g., HyClone CCM3 medium)

  • Use baffled flasks (e.g., 250-ml for optimization, 2-l for production)

  • Maintain cell density at approximately 2 × 10^6 cells/ml prior to infection

• Viral Infection:

  • Use recombinant baculovirus with a multiplicity of infection (MOI) of 2

  • Monitor cell viability using Trypan blue staining

  • Optimal harvest time is approximately 48 hours post-infection, when cell viability reaches around 40%

• Expression Monitoring:

  • Evaluate expression by Western blot using antibodies against the protein or associated tags (e.g., His₆ tag)

  • Use appropriate secondary antibodies and visualization methods (e.g., Alexa Fluor 680, infrared imaging)

• Harvest and Processing:

  • Collect cells by centrifugation (1,000 g for 15 min)

  • Perform all subsequent steps at 4°C to maintain protein integrity

Researchers should note that the expression level can vary based on virus quality, cell passage number, and culture conditions. It is advisable to optimize these parameters for each specific research setting.

What are the most effective methods for purifying PlGF-3 from Sf9 cell cultures?

Effective purification of PlGF-3 from Sf9 cell cultures involves several sequential steps:

• Membrane Preparation:

  • Resuspend harvested cells in lysis buffer (e.g., 150 mM NaCl, 50 mM Tris/HCl, pH 7.4)

  • Include protease inhibitors (e.g., 10 μM chymostatin, 10 μM leupeptin, 1 μM pepstatin A, 0.2 mM PMSF)

  • Disrupt cells using a high-pressure homogenizer (e.g., EmulsiFlex-C3, 4,000 psi, 4°C)

  • Clarify the homogenate by centrifugation

• Chromatographic Purification:

  • Apply proprietary chromatographic techniques as indicated in source materials

  • Consider affinity chromatography if using tagged protein constructs

  • Use size exclusion chromatography for final polishing steps

• Formulation and Storage:

  • Lyophilize purified PlGF-3 from a 0.2μm filtered solution in HCl

  • For reconstitution, use sterile 4mM HCl at a concentration not less than 100μg/ml

  • For long-term storage, add carrier protein (0.1% HSA or BSA) and store below -18°C

  • Avoid freeze-thaw cycles to maintain protein integrity

Purity assessment should be performed using SDS-PAGE, with expected purity greater than 95% . The addition of carrier proteins for long-term storage is crucial for maintaining the stability and activity of the purified protein.

How can researchers verify the functionality of purified PlGF-3?

Verification of PlGF-3 functionality involves several complementary approaches:

• Receptor Binding Assays:

  • Measure binding ability to recombinant human VEGF R1 (FLT1) using functional ELISA

  • The ED50 for functional PlGF-3 should be less than 3ng/ml

• Biological Activity Assessment:

  • Evaluate endothelial cell proliferation and migration in response to PlGF-3

  • Perform tube formation assays using human umbilical vein endothelial cells (HUVECs) to assess angiogenic potential

  • Compare activity with positive controls (e.g., VEGF-A) and negative controls

• Structural Integrity Analysis:

  • Confirm homodimeric structure using non-reducing SDS-PAGE

  • Verify glycosylation status using glycoprotein-specific staining or mass spectrometry

  • Assess protein folding using circular dichroism or other spectroscopic methods

• Downstream Signaling Evaluation:

  • Measure phosphorylation of VEGFR-1 and downstream kinases (e.g., ERK, Akt) in responsive cells

  • Monitor activation of angiogenesis-related gene expression using qPCR or RNA-seq

When evaluating biological activity, researchers should be aware that PlGF-3's effects may differ from those of other PlGF isoforms due to its lack of heparin-binding domains. Experimental designs should account for these potential differences when comparing across isoforms.

How does PlGF-3 compare functionally to other angiogenic factors?

PlGF-3 exhibits distinct functional characteristics compared to other angiogenic factors:

These distinctive properties make PlGF-3 a valuable tool for studying specialized aspects of angiogenesis and may offer therapeutic advantages by targeting pathological angiogenesis with potentially fewer side effects than broad VEGF inhibition.

What experimental models are most suitable for studying PlGF-3 functions?

Several experimental models are particularly well-suited for studying PlGF-3 functions:

• Cell Culture Models:

  • Human umbilical vein endothelial cells (HUVECs) for tube formation assays

  • Human retinal pigment epithelial (RPE) cells for studying ocular angiogenesis

  • Macrophage cultures for investigating inflammatory responses

  • VEGFR-1 expressing cell lines for receptor activation studies

• Ex Vivo Models:

  • Placental explant cultures for studying PlGF-3's role in placental development

  • Retinal explants for investigating neovascularization processes

  • Aortic ring assays for quantifying vessel sprouting

• In Vivo Models:

  • Targeted gene knockdown using siRNA in specific tissues

  • Transgenic mouse models with tissue-specific expression

  • Chorioallantoic membrane (CAM) assays for angiogenesis studies

  • Ischemia models (hindlimb, myocardial) for therapeutic potential assessment

• Disease Models:

  • Pre-eclampsia models for investigating placental insufficiency

  • Tumor xenograft models for studying cancer angiogenesis

  • Inflammatory disease models for examining PlGF's role in immune regulation

When designing experiments, researchers should consider that mice only express the equivalent of human PlGF-2, lacking the other isoforms present in humans . This species difference should be accounted for when translating findings between murine models and human applications.

How can PlGF-3 be used in angiogenesis and cancer research?

PlGF-3 offers several valuable applications in angiogenesis and cancer research:

• Mechanistic Studies:

  • Investigating VEGFR-1-specific signaling pathways without heparin-binding effects

  • Exploring the role of PlGF in tumor angiogenesis independently of VEGF

  • Studying the interactions between tumor cells and associated macrophages

• Biomarker Research:

  • Evaluating PlGF-3 as a potential biomarker for specific cancer types

  • Correlating PlGF-3 expression with tumor progression and treatment response

  • Developing isoform-specific detection methods for improved diagnostic accuracy

• Therapeutic Development:

  • Testing PlGF-3 inhibition as a potential anti-angiogenic therapy with potentially fewer side effects than VEGF inhibition

  • Investigating combination therapies targeting multiple angiogenic pathways

  • Using recombinant PlGF-3 to promote therapeutic angiogenesis in ischemic conditions

• Research Tools:

  • Using PlGF-3 as a specific VEGFR-1 agonist in experimental settings

  • Developing PlGF-3-based affinity reagents for receptor purification

  • Creating reporter systems to monitor PlGF-3-induced signaling

Research has shown circulating PlGF levels correlate with colorectal and renal cancers, as well as atherosclerosis and ischemic heart disease . The unique receptor specificity of PlGF-3 provides opportunities to develop more targeted interventions for these conditions.

What are the key differences in expression patterns between PlGF-3 and other isoforms?

The expression patterns of PlGF isoforms show significant tissue specificity and regulation:

• Tissue Distribution:

  • PlGF is primarily detected in the placenta, heart, lungs, thyroid, and adipose tissues

  • PlGF-3 shows particularly interesting distribution with very low levels in serum but relatively higher amounts in placental tissue

  • PlGF-3 in placental tissue appears prominently associated with cellular membranes

• Expression Levels:

  • In most serum samples collected during pregnancy, PlGF-3 was below the detection limit of 1.6 pg/mL, in contrast to more abundant PlGF-1 and PlGF-2

  • The differential expression suggests specialized roles for each isoform in specific tissues

• Species Differences:

  • Humans express four PlGF isoforms (PlGF-1–4)

  • Mice only express the equivalent of human PlGF-2, lacking the other isoforms

  • This species difference is crucial to consider when translating research findings

• Pathological Regulation:

  • Expression patterns of PlGF isoforms may change during disease states

  • While total PlGF levels correlate with certain cancers and cardiovascular conditions, isoform-specific alterations are still being investigated

Understanding these differential expression patterns provides insights into the specialized functions of each isoform and guides the selection of appropriate experimental models for studying their roles in normal physiology and disease.

How do the structural differences between PlGF isoforms affect their functional properties?

The structural differences between PlGF isoforms significantly impact their functional properties:

• Heparin-Binding Domains:

  • PlGF-2 contains a heparin-binding domain consisting of 21 basic amino acids at the carboxyl terminus

  • PlGF-3 lacks this heparin-binding domain

  • This difference affects interactions with cell surface proteoglycans and extracellular matrix components

• Receptor Interactions:

  • All PlGF isoforms bind to VEGFR-1/FLT1

  • PlGF-2 can additionally bind to neuropilin (NRP)-1 and -2 due to its heparin-binding domain

  • PlGF-3's receptor interactions are more restricted, focusing its signaling through VEGFR-1

• Protein Size and Glycosylation:

  • PlGF-1 is a glycosylated homodimer containing 2 x 131 amino acids (~34 kDa)

  • PlGF-3 is a glycosylated homodimer containing 2 x 203 amino acids (~58 kDa)

  • These differences in size and structure affect protein stability, half-life, and tissue penetration

• Secretion and Localization:

  • PlGF isoforms have unique secretion patterns

  • PlGF-3 shows strong association with cellular membranes in placental tissue

  • These localization differences can influence local concentration gradients and signaling outcomes

The functional consequences of these structural differences include altered tissue distribution, receptor activation kinetics, and biological responses, making each isoform uniquely suited for specific physiological contexts and experimental applications.

What methodological considerations should be made when comparing data across different PlGF isoforms?

When comparing data across different PlGF isoforms, researchers should consider several methodological factors:

• Isoform-Specific Detection:

  • Ensure antibodies and detection methods can distinguish between isoforms

  • Use isoform-specific immunoassays like the PlGF-3 specific DELFIA research immunoassay

  • Be aware of potential cross-reactivity in commercial detection kits

• Expression Systems:

  • Compare proteins produced in the same expression system when possible

  • Note that Sf9-produced proteins may have different glycosylation patterns than mammalian cell-produced proteins

  • Document complete details of the expression system used

• Functional Assays:

  • Use consistent functional assays when comparing isoforms

  • Account for the different receptor-binding profiles of each isoform

  • Include appropriate positive and negative controls

• Concentration Determination:

  • Use standardized methods for protein quantification

  • Account for differences in molecular weight when comparing molar concentrations

  • Consider the active concentration versus total protein concentration

• Statistical Analysis:

  • Use appropriate statistical methods for comparing potency and efficacy

  • Account for sample size limitations in rare isoforms like PlGF-3

  • Consider non-parametric tests when distribution assumptions are not met

What are the current technical challenges in PlGF-3 research?

Several technical challenges persist in PlGF-3 research that researchers should be aware of:

• Detection Limitations:

  • Low natural abundance of PlGF-3 in biological samples makes detection challenging

  • Most serum samples show PlGF-3 levels below detection limits (1.6 pg/mL)

  • Need for development of more sensitive detection methods

• Isoform Specificity:

  • Difficulty in generating truly isoform-specific antibodies

  • Challenges in distinguishing biological effects of different isoforms in mixed systems

  • Limited availability of validated isoform-specific reagents

• Recombinant Protein Quality:

  • Variations in glycosylation patterns between different expression systems

  • Potential for protein aggregation during purification and storage

  • Maintaining consistent dimer formation in recombinant preparations

• Functional Assays:

  • Standardization of biological activity assays across laboratories

  • Distinguishing direct effects from indirect effects mediated by other factors

  • Translating in vitro findings to in vivo significance

• Species Differences:

  • Mice only express the equivalent of human PlGF-2

  • Challenges in developing relevant animal models for PlGF-3-specific functions

  • Limitations in translating findings from animal models to human biology

Addressing these challenges requires multidisciplinary approaches combining advanced protein chemistry, sensitive analytical methods, and sophisticated biological assays.

How can researchers optimize PlGF-3 stability and activity in experimental settings?

Optimizing PlGF-3 stability and activity requires careful attention to several key factors:

• Reconstitution and Storage:

  • Reconstitute lyophilized PlGF-3 in sterile 4mM HCl at a concentration not less than 100μg/ml

  • For short-term storage (2-7 days), keep at 4°C

  • For long-term storage, store below -18°C with a carrier protein (0.1% HSA or BSA)

  • Avoid repeated freeze-thaw cycles

• Buffer Optimization:

  • When diluting from stock solution, choose buffers that maintain protein stability

  • Consider the impact of pH, ionic strength, and buffer components on protein structure

  • Test stability in your specific experimental buffer before conducting critical experiments

• Handling Practices:

  • Minimize exposure to room temperature

  • Avoid vigorous vortexing or shaking that can cause protein denaturation

  • Use low-protein-binding plasticware for dilute solutions

• Activity Preservation:

  • Include appropriate protease inhibitors in experimental buffers

  • Consider the addition of reducing agents if disulfide bond integrity is a concern

  • Verify activity through functional assays before critical experiments

• Documentation and Standardization:

  • Maintain detailed records of handling procedures

  • Use consistent protocols across experiments

  • Include activity controls in each experimental run

By implementing these optimization strategies, researchers can enhance experimental reproducibility and maximize the utility of recombinant PlGF-3 in their studies.

What emerging techniques are advancing PlGF-3 research?

Several emerging techniques are significantly advancing PlGF-3 research:

• Single-Cell Analysis:

  • Single-cell RNA sequencing to identify cell populations expressing specific PlGF isoforms

  • Single-cell protein analysis to detect isoform-specific expression patterns

  • Spatial transcriptomics to map PlGF-3 expression in tissue contexts

• Advanced Protein Engineering:

  • Site-specific labeling for tracking PlGF-3 in cellular systems

  • Creation of chimeric proteins to dissect domain-specific functions

  • Development of stabilized variants with enhanced half-life

• High-Sensitivity Detection Methods:

  • Digital ELISA platforms with femtomolar sensitivity

  • Mass spectrometry-based approaches for isoform-specific quantification

  • Proximity ligation assays for detecting protein-protein interactions

• Computational Approaches:

  • Structural modeling of PlGF-3/receptor interactions

  • Systems biology approaches to map PlGF-3-specific signaling networks

  • Machine learning algorithms to predict PlGF-3 functions from large datasets

• Gene Editing Technologies:

  • CRISPR/Cas9-based approaches for isoform-specific knockouts

  • Precise genome editing to create humanized animal models expressing PlGF-3

  • Cell line engineering for controlled expression of specific isoforms

These emerging techniques will likely address many current limitations in PlGF-3 research and provide deeper insights into its specialized functions in development, homeostasis, and disease.