VTCN1 Human, Sf9

What is VTCN1 and what is its function in the immune system?

VTCN1 (V-set domain-containing T-cell activation inhibitor 1) belongs to the B7 costimulatory protein family and plays a crucial role in immune regulation. It is located on the surface of antigen-presenting cells and interacts with receptors on T cells to inhibit T-cell activation and proliferation . VTCN1 functions as a negative regulator of immune responses by suppressing T-cell activity.

For investigating VTCN1's immunomodulatory function, researchers typically employ:

• Co-culture assays with T cells and antigen-presenting cells expressing VTCN1

• Flow cytometry to assess T-cell activation markers

• Cytokine production assays after VTCN1 engagement

• Functional blocking studies using anti-VTCN1 antibodies

High expression of VTCN1 has been associated with tumor progression, likely by suppressing anti-tumor immune responses and promoting tumor immune evasion .

What are the structural characteristics of VTCN1 Human, Sf9?

VTCN1 Human produced in Sf9 cells exhibits distinct structural properties important for research applications:

• Single glycosylated polypeptide chain containing 244 amino acids (residues 25-259)

• Theoretical molecular mass of 26.9kDa

• Appears at approximately 28-40kDa on SDS-PAGE due to glycosylation

• Contains a 9-amino acid His-tag at the C-terminus for purification purposes

• Formulated in Phosphate Buffered Saline (pH 7.4) with 10% glycerol

The protein contains an immunoglobulin V-set domain characteristic of the B7 family, which mediates receptor interactions. The discrepancy between theoretical molecular weight (26.9kDa) and observed SDS-PAGE migration (28-40kDa) reflects heterogeneous glycosylation in the Sf9 expression system .

To characterize VTCN1 structure, researchers commonly employ:

• SDS-PAGE for purity assessment and molecular weight determination

• Western blotting for identity confirmation

• Mass spectrometry for detailed molecular weight and glycosylation analysis

• Circular dichroism for secondary structure assessment

How is VTCN1 Human recombinant protein produced in Sf9 cells?

Production of VTCN1 Human recombinant protein in Sf9 Baculovirus cells follows a multi-step process:

• Gene cloning: The human VTCN1 gene sequence (encoding amino acids 25-259) is inserted into a baculovirus transfer vector with a C-terminal His-tag.

• Baculovirus generation: The recombinant vector is co-transfected with baculovirus DNA into insect cells to produce recombinant baculovirus particles.

• Expression: Sf9 insect cells are infected with the recombinant baculovirus carrying the VTCN1 gene, driving protein expression .

• Purification: The expressed protein with its C-terminal His-tag is purified using proprietary chromatographic techniques, likely involving immobilized metal affinity chromatography (IMAC) .

This process yields a single, glycosylated polypeptide chain containing 244 amino acids with a molecular mass of 26.9kDa (appearing as 28-40kDa on SDS-PAGE due to glycosylation) .

The Sf9-Baculovirus system offers several advantages for VTCN1 production:

• Capacity for post-translational modifications, particularly glycosylation

• Higher yield compared to mammalian expression systems

• Ability to express complex proteins with proper folding

• Scalability for larger production requirements

What are the storage and stability conditions for VTCN1 Human, Sf9?

Proper storage of VTCN1 Human, Sf9 is critical for maintaining protein functionality in research applications:

Short-term storage (2-4 weeks):

• Store at 4°C

Long-term storage:

• Store frozen at -20°C

• For extended periods, add a carrier protein (0.1% HSA or BSA) to enhance stability

The standard formulation consists of VTCN1 protein solution (0.25mg/ml) in Phosphate Buffered Saline (pH 7.4) with 10% glycerol . The glycerol component helps prevent freeze-thaw damage.

Critical handling recommendations include:

• Avoiding multiple freeze-thaw cycles which can lead to protein denaturation

• Aliquoting the protein solution before freezing to minimize freeze-thaw cycles

• Thawing frozen protein slowly on ice to minimize thermal stress

• Performing stability assessments using SDS-PAGE and functional assays

Following these storage protocols helps maintain structural integrity and biological activity of VTCN1 for reliable experimental outcomes.

What techniques can be used to validate the purity and functionality of VTCN1 Human, Sf9?

Comprehensive validation of VTCN1 Human, Sf9 requires multiple complementary techniques assessing both purity and biological activity:

Purity Assessment:

• SDS-PAGE: Should show a predominant band at 28-40kDa with purity greater than 90.0%

• Size Exclusion Chromatography (SEC): To detect aggregates and evaluate oligomeric state

• Western Blotting: Using anti-VTCN1 or anti-His-tag antibodies for identity confirmation

Protein Characterization:

• Mass Spectrometry: For accurate molecular weight and post-translational modification analysis

• N-terminal Sequencing: To confirm correct protein processing

• Peptide Mapping: To verify sequence coverage and identify modifications

Functional Validation:

• Binding Assays:

  • ELISA with known binding partners

  • Surface Plasmon Resonance (SPR) for binding kinetics

  • Bio-Layer Interferometry (BLI) for real-time interaction analysis

• Cell-Based Assays:

  • T-cell proliferation inhibition assay

  • Cytokine secretion measurement

  • Flow cytometry for cell surface binding assessment

A methodical validation approach should proceed from purity assessment to structural characterization and finally functional testing, documenting all results with appropriate controls to ensure research reliability.

How does VTCN1 influence cellular signaling pathways in tumor progression?

VTCN1 regulates multiple signaling pathways implicated in tumor progression, as revealed by detailed molecular studies:

MAPK/ERK1/2 Pathway:

• VTCN1 knockdown activates the MAPK/ERK1/2 pathway

• RNA-seq data showed decreased MAPK transcripts but, paradoxically, increased phospho-MAPK (pMAPK) protein levels after VTCN1 knockdown

• This pathway promotes invasion in cell models of extravillous trophoblast (EVT) lineage, suggesting a mechanism for VTCN1's involvement in regulating cellular invasion

JAK/STAT Pathway:

• VTCN1 knockdown increases both STAT1 transcripts and phospho-STAT1 (pSTAT1) protein levels

• Activation of this pathway correlates with enhanced invasive properties

• Conversely, STAT1 inhibition limits invasion in trophoblast model cell lines

Pro-Invasion Markers:

• VTCN1 suppression upregulates:

  • Integrin Subunit Alpha 5 (ITGA5)

  • Matrix metalloproteinase-12 (MMP-12)

  • These markers drive enhanced invasive capacity

To investigate these mechanisms, researchers typically employ:

• RNA interference or CRISPR/Cas9 gene editing to modulate VTCN1 expression

• RNA-seq and protein phosphorylation analyses to identify affected pathways

• Invasion assays to correlate signaling changes with functional outcomes

• Pathway-specific inhibitors to confirm causality

These findings suggest VTCN1 normally constrains cellular invasion pathways, which may explain how its aberrant expression could promote tumor progression through altered cell behavior.

What role does VTCN1 play in trophoblast development and placental function?

VTCN1 serves as a critical regulator of trophoblast development and placental function through multiple mechanisms:

Trophoblast Invasion Regulation:

• VTCN1 knockdown activates signaling pathways (MAPK/ERK1/2 and JAK/STAT) that promote trophoblast invasion

• Enhanced expression of pro-invasion markers (ITGA5 and MMP-12) occurs when VTCN1 is suppressed

• These findings position VTCN1 as a negative regulator of trophoblast invasion, a process critical for proper placentation

Syncytialization Control:

• VTCN1 knockdown increases expression of IFITM1 (IFN-induced transmembrane protein 1)

• IFITM1 impairs syncytin-mediated cell fusion, a key process in syncytiotrophoblast formation

• This explains the observed reduction in syncytiotrophoblast (STB) formation when VTCN1 is knocked down

Immune Regulation at Maternal-Fetal Interface:

• VTCN1 knockdown increases expression of classical MHC class I molecules (HLA-A, HLA-B, HLA-C) while HLA-G remains relatively unchanged

• This shift in MHC expression could affect maternal immune tolerance of fetal tissues

• VTCN1 may contribute to establishing the immunologically privileged status of the developing placenta

These functions suggest VTCN1 helps orchestrate the complex balance between trophoblast invasion, syncytialization, and immune privilege required for successful placental development and function.

How does VTCN1 knock-down affect MHC class I expression?

VTCN1 exerts differential regulatory effects on MHC class I molecules, potentially influencing immune recognition at the maternal-fetal interface:

Classical MHC Class I (HLA-A, HLA-B, HLA-C):

• RNA-seq analyses revealed significant increases in mRNA levels of classical MHC class I molecules following VTCN1 suppression

• RT-PCR validation confirmed increased transcript levels on days 5-6 of BAP treatment (days 2-3 post-VTCN1 knockdown)

• Protein expression increases were verified by western blotting, immunocytochemistry, and flow cytometry

Non-Classical MHC Class I (HLA-G):

• HLA-G transcripts remained relatively unchanged following VTCN1 knockdown

• Protein levels of HLA-G were similarly unaffected

• This is biologically significant as HLA-G is a marker for extravillous trophoblast (EVT) cells and crucial for maternal-fetal immune tolerance

This differential regulation has important implications:

• Increased classical MHC I expression could potentially enhance recognition by maternal immune cells

• Maintained HLA-G expression would preserve some immune-privileged properties

• The balance between these MHC molecules affects cellular immunogenicity

These findings position VTCN1 as a selective regulator of MHC class I expression, with potential implications for immune evasion mechanisms in both placental development and tumor biology.

What is the relationship between VTCN1 and IFITM1 expression in trophoblast cells?

VTCN1 and IFITM1 (IFN-induced transmembrane protein 1) exhibit a significant regulatory relationship in trophoblast development with functional consequences:

Expression Relationship:

• RNA-seq data demonstrated that VTCN1 knockdown significantly increases IFITM1 transcript levels

• Protein-level validation by Western blotting confirmed this upregulation

• IFITM1 protein levels increased as syncytialization decreased when VTCN1 was knocked down in BAP-treated cells

Functional Significance:

• IFITM1 belongs to a family of proteins (IFITM1, -2, and -3) that prevent viral membrane fusion with cells

• IFITM1 specifically impairs syncytin-mediated cell fusion, which is essential for syncytiotrophoblast formation

• Syncytialization is a fundamental process in trophoblast development

Mechanism and Implications:

• VTCN1 appears to normally suppress IFITM1 expression

• When VTCN1 is knocked down, IFITM1 expression increases

• Elevated IFITM1 inhibits syncytin-mediated cell fusion

• This leads to reduced syncytiotrophoblast formation

This regulatory relationship provides a molecular mechanism for how VTCN1 influences trophoblast differentiation. The VTCN1-IFITM1 axis represents a novel pathway controlling cell fusion in placental development, with potential implications for placental disorders characterized by abnormal syncytialization.

How can CRISPR/Cas9 be used for studying VTCN1 function?

CRISPR/Cas9 genome editing offers powerful approaches for investigating VTCN1 function through precise genetic manipulation:

CRISPR/Cas9 Strategy for VTCN1 Knockout:

• Guide RNA Design: Study employed two sgRNAs (upstream and downstream) targeting specific loci within the VTCN1 gene

• Complete knockout: CRISPR/Cas9 was used to generate VTCN1-/- human embryonic stem cells (hESCs)

• This approach allows for studying complete loss of VTCN1 function, contrasting with the partial reduction achieved with siRNA knockdown

Methodological Workflow:

• Guide RNA Design:

  • Select target sequences within VTCN1 with minimal off-target effects

  • Design complementary sgRNAs binding to these regions

• Delivery System:

  • Transfect cells with plasmids encoding Cas9 and sgRNAs

  • Alternatively, use ribonucleoprotein (RNP) complexes for transient editing

• Clone Selection and Validation:

  • Screen for successful editing using genomic PCR and sequencing

  • Confirm VTCN1 knockout at protein level via Western blotting

• Functional Analysis:

  • Compare wild-type and VTCN1-/- cells using:

    • Transcriptome analysis (RNA-seq)

    • Protein expression profiling

    • Cell signaling pathway assessment

    • Functional assays relevant to the cell type

Advanced CRISPR Applications for VTCN1 Studies:

• CRISPR activation (CRISPRa) for upregulating VTCN1 expression

• CRISPR interference (CRISPRi) for targeted repression

• Knock-in of tagged VTCN1 for protein localization studies

• Introduction of specific mutations to study structure-function relationships

CRISPR/Cas9 approaches overcome limitations of traditional knockdown methods, providing stable genetic modifications that enable comprehensive investigation of VTCN1's biological functions across different cellular contexts.

How does glycosylation affect VTCN1 function, and how does Sf9 expression impact glycosylation patterns?

Glycosylation significantly influences VTCN1 function, and the Sf9 expression system imparts distinct glycosylation patterns with important research implications:

Role of Glycosylation in VTCN1 Function:

• Influences protein folding and structural stability

• Affects receptor binding affinity and specificity

• Impacts immunogenicity and immune system interactions

• Contributes to protein half-life and clearance in vivo

Sf9-Specific Glycosylation Characteristics:

• Sf9 cells primarily produce high-mannose type N-glycans, unlike the complex N-glycans in mammalian cells

• The apparent molecular weight of VTCN1 Human, Sf9 on SDS-PAGE (28-40kDa) versus theoretical mass (26.9kDa) indicates significant glycosylation

• The variable band appearance (28-40kDa) suggests heterogeneous glycosylation

• Sf9 cells lack the ability to produce mammalian-like sialylated complex glycans

Functional Implications:

• Altered receptor binding kinetics possible due to glycan differences

• Potentially different immunomodulatory properties compared to mammalian-expressed VTCN1

• May affect protein stability and solubility

• Could influence results in certain experimental systems

Methodological Approaches for Assessment:

• Glycosylation Analysis:

  • Mass spectrometry to characterize glycan structures

  • Lectin binding assays to profile glycan types

  • Enzymatic deglycosylation to assess contribution to molecular weight

• Comparative Functional Studies:

  • Side-by-side testing with mammalian-expressed VTCN1

  • Binding kinetics analysis with receptors

  • Cell-based immunomodulatory assays

Understanding these glycosylation differences is crucial when interpreting experimental results and evaluating whether Sf9-expressed VTCN1 is appropriate for specific research applications.

What experimental approaches are recommended for investigating VTCN1's role in immune regulation?

Comprehensive investigation of VTCN1's immune regulatory functions requires multi-faceted experimental approaches spanning molecular, cellular, and in vivo techniques:

Molecular and Biochemical Approaches:

• Protein-Protein Interaction Studies:

  • Surface Plasmon Resonance (SPR) to identify binding partners and measure kinetics

  • Co-immunoprecipitation to detect protein complexes

  • Proximity ligation assays to visualize interactions in situ

  • FRET/BRET for real-time interaction dynamics

• Structural Biology:

  • X-ray crystallography or cryo-EM of VTCN1 alone and in complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Mutagenesis studies to identify critical functional residues

Cellular Immunology Approaches:

• T Cell Functional Assays:

  • Proliferation assays using CFSE dilution or BrdU incorporation

  • Cytokine production measurement by ELISA, ELISpot, or intracellular staining

  • Cytotoxicity assays to assess killer T cell function

• Antigen Presenting Cell (APC) Studies:

  • Flow cytometry to analyze VTCN1 expression on different APC subsets

  • Mixed lymphocyte reactions with VTCN1-expressing APCs

  • Dendritic cell maturation and function assays

• Gene Modification Approaches:

  • CRISPR/Cas9 knockout of VTCN1 in relevant cell types

  • siRNA knockdown for temporal control of expression

  • Overexpression studies to assess gain-of-function effects

Advanced Systems Approaches:

• Transcriptomics and Proteomics:

  • RNA-seq of immune cells after VTCN1 engagement

  • Phosphoproteomics to map signaling cascades

  • Single-cell RNA-seq to assess heterogeneity in response

• In Vivo Models:

  • VTCN1 knockout mouse models

  • Humanized mouse models with human immune components

  • Tumor models to assess VTCN1's role in anti-tumor immunity

These approaches provide complementary insights into VTCN1's multifaceted roles in immune regulation and can help identify potential therapeutic targets for immunomodulation in cancer and autoimmune conditions.

How does VTCN1 Human, Sf9 compare to VTCN1 produced in other expression systems?

Comparing VTCN1 Human produced in different expression systems reveals important differences that can affect experimental outcomes:

Comparative Expression System Analysis:

Mammalian Expression Systems (e.g., HEK293):

• More complex and human-like glycosylation patterns

• Potentially more physiologically relevant post-translational modifications

• Generally lower yield than insect cell systems

• Higher production costs

• Slower production timeline

Sf9 Baculovirus System:

• Simpler glycosylation patterns (primarily high-mannose type)

• Higher expression yields

• More cost-effective production

• Faster production timeline

• Good protein folding capacity for complex proteins

Bacterial Expression Systems (e.g., E. coli):

• No glycosylation

• May require refolding from inclusion bodies

• Highest yield and lowest cost

• May lack critical post-translational modifications for function

• Fastest production timeline

Drawing parallels from AAV production comparability studies, "demonstration of comparability does not necessarily mean that the quality attributes of the pre-change and post-change product are identical" . This principle applies to VTCN1 production across different expression systems.

For many research applications, VTCN1 Human, Sf9 offers a good balance of proper folding, moderate glycosylation, and cost-effective production, but researchers should select the expression system based on their specific experimental requirements.

What are the challenges in producing functional VTCN1 Human, Sf9 for research applications?

Producing functional VTCN1 Human in Sf9 cells presents several technical challenges that researchers must address:

Expression System Limitations:

• Sf9 cells produce different glycosylation patterns compared to mammalian cells, which may affect VTCN1 function

• The molecular weight heterogeneity observed on SDS-PAGE (28-40kDa vs. theoretical 26.9kDa) indicates variable glycosylation

• While Sf9 expression offers higher yields than mammalian systems, it may not perfectly recapitulate all post-translational modifications

Protein Stability Considerations:

• VTCN1 Human, Sf9 requires specific storage conditions (4°C for short-term, -20°C with carrier protein for long-term)

• Multiple freeze-thaw cycles compromise protein integrity and function

• Formulation with 10% glycerol helps maintain stability but may interfere with some applications

Functional Verification Challenges:

• Confirming that the recombinant protein retains native binding properties requires specialized assays

• The C-terminal His-tag may potentially interfere with some protein-protein interactions

• Comparative activity testing against mammalian-expressed VTCN1 is necessary to validate functionality

Methodological Solutions:

• Optimizing Expression Parameters:

  • Fine-tuning infection conditions (MOI, harvest time)

  • Testing different signal sequences or fusion partners

  • Evaluating various purification strategies to enhance yield and purity

• Enhancing Stability:

  • Screening buffer compositions to identify optimal formulation

  • Adding stabilizing agents (carrier proteins, specific salts)

  • Developing lyophilized formulations for long-term storage

• Comprehensive Validation Strategy:

  • Binding assays with known interaction partners

  • Cell-based assays to confirm immunomodulatory activity

  • Comparative studies with mammalian-expressed VTCN1

Addressing these challenges requires careful optimization and validation to ensure that VTCN1 Human, Sf9 maintains structural integrity and biological activity for reliable research applications.