PSG1 Human, Sf9

What is human PSG1 and what is its biological significance?

Human Pregnancy-Specific Glycoprotein 1 (PSG1) belongs to a family of secreted proteins produced by the placenta during pregnancy that play critical roles in pregnancy success. PSG1 has been demonstrated to induce secretion of anti-inflammatory cytokines from monocytes and macrophages in vitro, including interleukin-10 (IL-10), IL-6, and transforming growth factor-β1 (TGF-β1) . This immunomodulatory activity contributes to shifting the maternal immune system from a predominantly TH1 response to a TH2 response, which is considered more compatible with successful pregnancy . The induction of these anti-inflammatory cytokines by PSGs supports the hypothesis that these glycoproteins have an essential role in regulating maternal immune responses in species with hemochorial placentation .

How does human PSG1 compare to mouse PSGs in terms of receptor interactions?

Human and mouse PSGs exhibit significant differences in receptor interactions despite having similar immunomodulatory functions. Mouse PSG17 has been identified to bind to the integrin-associated CD9 receptor on macrophages, and this interaction is necessary for the induction of anti-inflammatory cytokines . The CD9 receptor binds specifically to the N1 domain of both mouse Psg17 and Psg19 .

In contrast, human PSGs operate through a different mechanism. The search results explicitly state that "unlike mouse Psg17, human PSG do not require CD9 to induce cytokine production from mouse macrophages" . No receptor for human PSG has been identified yet, indicating that human PSGs likely utilize an alternative receptor or signaling pathway to exert their immunomodulatory effects . This difference highlights the evolutionary divergence in molecular mechanisms while preserving similar biological functions.

Why are Sf9 cells particularly suitable for expressing recombinant PSG1?

Sf9 cells (derived from Spodoptera frugiperda) are highly advantageous for expressing recombinant human PSG1 for several reasons. These insect cells, when used with the baculovirus expression system, can produce large quantities of properly folded eukaryotic proteins with many post-translational modifications similar to mammalian cells .

The baculovirus-Sf9 system allows for proper glycosylation (though patterns differ from human cells), disulfide bond formation, and protein secretion - all critical for glycoproteins like PSG1. Additionally, Sf9 cells can be grown in serum-free medium (SF900 II), which simplifies downstream purification processes . The system also accommodates large protein inserts and can be scaled up efficiently, as demonstrated in the search results where large-scale expression was carried out in one-liter spinner flasks . Finally, the ability to add purification tags (like the V5/His tag mentioned in the results) facilitates protein detection and purification without interfering with the functional domains .

What is the optimal protocol for expressing human PSG1 in Sf9 cells?

Based on the search results, the optimal protocol for expressing human PSG1 in Sf9 cells involves a comprehensive multi-step process:

• Vector Construction: The human PSG1a open reading frame should be amplified by PCR from an existing cDNA clone and cloned into an appropriate baculovirus transfer vector such as pBlueBac4.5V5/His. Importantly, the construct should be designed with the V5/His tag at the C-terminus "to prevent interference of the tag with putative Psg functional domains at the N terminus" .

• Recombinant Baculovirus Generation: Following transfection, recombinant baculoviruses should be isolated through plaque assay. Blue plaques (representing sites of recombinant viral infection) should be picked and used to infect fresh Sf9 cells. Cultures should be observed for signs of recombinant-only viral infection (absence of occlusion bodies, OCC–) .

• Viral Stock Preparation: Sequential viral stocks should be generated:

  • P1 stock: Initial viral stock from OCC– wells, confirmed by PCR analysis to be free of wild-type baculovirus

  • P2 stock: Small-scale high-titer stock generated from P1

  • P3 stock: Large-scale high-titer stock generated from P2

  • Final titer determination through plaque assay

• Expression Optimization: Small-scale protein expression trials should be conducted to establish optimal multiplicity of infection (MOI) and time course. The search results indicate that an MOI of 3 phage particles per Sf9 cell with a harvest time of 5 days in SF900 II medium was optimal .

• Large-scale Production: Once optimized, scale up to one-liter spinner flasks using the determined MOI and time course in serum-free medium .

• Purification: After the culture period, cells should be removed by centrifugation, and the recombinant protein purified from the medium using an appropriate method such as the Xpress Protein Purification system .

This methodical approach ensures consistent and efficient production of functional recombinant human PSG1.

What signaling pathways are activated by PSG1, and how can they be experimentally investigated?

While the search results don't provide complete details about human PSG1 signaling pathways specifically, insights can be drawn from studies on mouse PSG17, which has similar functions in inducing anti-inflammatory cytokines:

• Cyclooxygenase 2 (COX-2) Pathway: Inhibition of COX-2 significantly reduces PSG17N-mediated increases in IL-10 and IL-6, suggesting this enzyme plays a crucial role in PSG-induced cytokine production .

• cAMP-PKA Pathway: Cyclic adenosine monophosphate-dependent protein kinase A (PKA) is involved in the up-regulation of IL-10 and IL-6, but not required for TGF-β1 induction .

• PKC Pathway: Protein kinase C (PKC) inhibition reduces PSG17-mediated induction of TGF-β1, IL-6, and IL-10 significantly, indicating PKC's importance in multiple cytokine induction pathways .

These pathways can be experimentally investigated through several approaches:

• Pharmacological Inhibition: Using specific inhibitors of signaling components (COX-2 inhibitors, PKA inhibitors like H-89, PKC inhibitors like staurosporine) to determine their role in PSG1-mediated cytokine induction.

• Phosphorylation Assays: Detecting activation of signaling molecules through phospho-specific antibodies after PSG1 treatment.

• Gene Expression Analysis: RNA-seq or qPCR to identify genes regulated by PSG1 and infer activated pathways.

• Protein-Protein Interaction Studies: Co-immunoprecipitation or proximity ligation assays to identify proteins interacting with PSG1 or its receptor(s).

• Reporter Assays: Using cells transfected with pathway-specific reporter constructs to measure pathway activation after PSG1 treatment.

Each of these methodologies helps delineate the complex signaling mechanisms through which PSG1 exerts its immunomodulatory effects.

What are critical quality control parameters for recombinant PSG1 produced in Sf9 cells?

Ensuring the quality of recombinant PSG1 produced in Sf9 cells requires comprehensive quality control measures:

These quality control parameters ensure that the recombinant PSG1 accurately represents the native protein's properties and is suitable for downstream research applications.

How can recombinant PSG1 be used to study immune modulation during pregnancy?

Recombinant PSG1 expressed in Sf9 cells provides a valuable tool for investigating immune modulation during pregnancy through multiple experimental approaches:

• In vitro Cytokine Induction Studies:

  • Treating primary monocytes, macrophages, or dendritic cells with purified recombinant PSG1 at physiologically relevant concentrations

  • Measuring secretion of anti-inflammatory cytokines including IL-10, IL-6, PGE2, and TGF-β1 using ELISA or multiplex assays

  • Analyzing dose-response relationships and kinetics of cytokine induction

• Immune Cell Phenotype Analysis:

  • Flow cytometry to assess changes in activation markers, co-stimulatory molecules, and intracellular cytokines in PSG1-treated immune cells

  • Investigation of PSG1's effects on macrophage polarization (M1 vs. M2)

  • Analysis of PSG1's impact on T cell differentiation (Th1/Th2/Th17/Treg balance)

• Receptor Identification Studies:

  • Cross-linking experiments with labeled PSG1 followed by mass spectrometry

  • Cell-based binding assays using fluorescently-labeled PSG1

  • Affinity purification with immobilized PSG1 to identify binding partners

  • Comparative studies between species to understand divergent receptor usage

• Signaling Pathway Analysis:

  • Pharmacological inhibition of specific pathways (COX-2, PKA, PKC) to determine their roles in PSG1-mediated effects

  • Western blot analysis of phosphorylation events in key signaling molecules

  • Transcriptome analysis to identify genes regulated by PSG1 treatment

• Ex vivo Models:

  • Using decidual tissue explants to study PSG1's effects in a more physiologically relevant context

  • Co-culture systems with trophoblasts and immune cells to model maternal-fetal interface

These methodologies can provide comprehensive insights into PSG1's role in promoting maternal-fetal immune tolerance during pregnancy.

What experimental approaches can determine the structure-function relationship of PSG1?

Understanding the structure-function relationship of PSG1 requires a multifaceted experimental approach:

These complementary approaches can provide comprehensive insights into how PSG1's structural elements contribute to its immunomodulatory functions and receptor specificity.

How can PSG1-CD9 interaction studies improve our understanding of species-specific mechanisms?

Although human PSG1 does not interact with CD9 (unlike mouse PSG17), comparative studies of PSG-receptor interactions across species can provide valuable insights:

• Evolutionary Analysis of Receptor Specificity:

  • The search results clearly state that "human PSG do not require CD9 to induce cytokine production from mouse macrophages"

  • Comparative experiments with human PSG1 and mouse PSG17 on both wild-type and CD9-deficient macrophages

  • Analysis of sequence conservation in N-terminal domains that mediate receptor binding

• Identification of the Human PSG1 Receptor:

  • Cross-species receptor screening to identify the human PSG1 receptor

  • Comparison of downstream signaling pathways activated by human PSG1 and mouse PSG17

  • Investigation of whether convergent evolution has led to similar functions through different receptor systems

• Structural Basis for Receptor Specificity:

  • Crystallography of mouse PSG17-CD9 complexes versus human PSG1 with its receptor

  • Identification of key residues that determine receptor binding specificity

  • Engineering of mouse PSG17 to bind human receptors or human PSG1 to bind CD9

• Functional Conservation Testing:

  • The search results indicate that "the ability of PSG17 to induce anti-inflammatory cytokines parallels that of members of the human PSG family, albeit human and murine PSGs use different receptors"

  • Comparative analysis of cytokine profiles induced by human and mouse PSGs

  • Investigation of whether the same anti-inflammatory pathways are activated despite different receptor usage

• Physiological Relevance in Different Species:

  • In vivo studies comparing the effects of species-specific PSGs in mouse models

  • Analysis of PSG expression patterns during pregnancy in different species

  • Correlation of PSG functions with placentation types across mammalian species

These studies would illuminate how different molecular mechanisms evolved to fulfill similar immunomodulatory functions critical for successful pregnancy across species with hemochorial placentation.

What are common pitfalls in PSG1 expression in Sf9 cells and how can they be resolved?

Researchers may encounter several challenges when expressing PSG1 in Sf9 cells:

• Low Expression Levels:

  • Problem: Insufficient protein yield despite successful infection

  • Solutions:

    • Optimize MOI through systematic testing (the search results indicate an MOI of 3 was optimal)

    • Adjust harvest time (5 days was optimal in the reported protocol)

    • Verify baculovirus titer and viability

    • Consider codon optimization for Sf9 cells

    • Evaluate different signal sequences for improved secretion

• Protein Degradation:

  • Problem: Detection of multiple bands or fragments in Western blot

  • Solutions:

    • Add protease inhibitors to culture medium

    • Harvest at earlier time points

    • Reduce culture temperature to 27°C

    • Test different buffer compositions during purification

    • Consider intracellular retention versus secretion strategies

• Improper Glycosylation:

  • Problem: Heterogeneous glycosylation patterns affecting protein function

  • Solutions:

    • Use tunicamycin to produce non-glycosylated protein for comparison

    • Engineer mutations to remove N-glycosylation sites

    • Consider alternative expression systems for mammalian-type glycosylation

    • Perform enzymatic deglycosylation post-purification

• Protein Aggregation:

  • Problem: Formation of protein aggregates during expression or purification

  • Solutions:

    • Optimize buffer conditions (pH, salt concentration)

    • Add stabilizing agents (glycerol, arginine)

    • Reduce protein concentration during purification

    • Implement size exclusion chromatography as a final purification step

• Tag Interference:

  • Problem: Purification tag affecting protein folding or function

  • Solutions:

    • Use a C-terminal tag as mentioned in the search results to avoid interference with N-terminal functional domains

    • Include a cleavable linker between the protein and tag

    • Compare tagged and untagged versions in functional assays

Addressing these challenges systematically can significantly improve the yield and quality of recombinant PSG1 expressed in Sf9 cells.

How can the baculovirus generation and amplification process be optimized?

Successful expression of PSG1 in Sf9 cells depends heavily on the quality of recombinant baculovirus. Based on the search results, several key optimization strategies can be implemented:

• Recombinant Virus Isolation:

  • Perform careful plaque assay to identify and isolate blue plaques representing recombinant virus

  • Verify the absence of occlusion bodies (OCC–), which indicates successful recombination

  • Conduct PCR analysis to confirm the absence of wild-type baculovirus contamination

• Sequential Viral Stock Generation:

  • Follow the systematic approach outlined in the search results:

    • P1 stock: Initial viral stock from confirmed recombinant-only infections

    • P2 stock: Small-scale high-titer stock from P1

    • P3 stock: Large-scale high-titer stock from P2

  • This sequential amplification ensures high-quality, high-titer viral stocks

• Viral Titer Determination:

  • Accurately determine viral titer using plaque assay

  • Consider alternative methods like qPCR or flow cytometry for rapid titer estimation

  • Maintain consistent titer determination methods throughout the process

• Storage and Stability:

  • Store viral stocks in small aliquots at -80°C to avoid repeated freeze-thaw cycles

  • Add 5-10% FBS as a stabilizer for long-term storage

  • Validate viral stability through titer determination after storage periods

• Infection Parameters:

  • Optimize cell density at infection (typically 1-2 × 10^6 cells/mL)

  • Use cells in mid-log phase growth for infection

  • Ensure uniform virus distribution through gentle agitation

  • Consider low-MOI amplification strategies for viral stock production

• Quality Control Checkpoints:

  • Implement visual inspection for signs of infection

  • Use control infections to verify viral activity

  • Monitor protein expression in small-scale cultures before large-scale production

Following these optimization strategies can significantly improve the consistency and efficiency of the baculovirus expression system for PSG1 production.

What are the optimal purification strategies for recombinant PSG1 from Sf9 culture media?

Purifying recombinant PSG1 from Sf9 culture media requires careful consideration of protein properties and downstream applications:

• Initial Processing:

  • Harvest culture at the optimal time point (5 days post-infection as mentioned in the search results)

  • Remove cells by centrifugation (1,000 × g for 10 minutes)

  • Filter the supernatant through a 0.22 μm filter to remove cell debris

  • Consider concentration using tangential flow filtration for large volumes

• Affinity Chromatography:

  • Utilize the His-tag for immobilized metal affinity chromatography (IMAC)

  • The search results mention using the Xpress Protein Purification system

  • Optimize binding conditions (pH, imidazole concentration) to reduce non-specific binding

  • Consider anti-V5 antibody affinity columns as an alternative approach

• Secondary Purification Steps:

  • Ion exchange chromatography based on PSG1's theoretical isoelectric point

  • Size exclusion chromatography to remove aggregates and provide buffer exchange

  • Hydrophobic interaction chromatography for removing structurally similar contaminants

• Buffer Optimization:

  • Test different buffer systems for optimal stability (phosphate, HEPES, Tris)

  • Include stabilizing agents (glycerol, sucrose, BSA) for long-term storage

  • Determine optimal pH range for stability (typically pH 7.0-8.0 for glycoproteins)

  • Consider adding specific protease inhibitors if degradation occurs

• Tag Removal Considerations:

  • If required for functional studies, remove the V5/His tag using specific proteases

  • Re-purify to separate the cleaved protein from the tag and protease

  • Compare activity of tagged versus untagged protein

• Quality Control Testing:

  • SDS-PAGE and Western blot to verify purity and identity

  • Mass spectrometry for accurate molecular weight determination

  • Glycosylation analysis using specialized staining or enzymatic treatments

  • Functional testing through cytokine induction assays

These purification strategies can be adapted based on PSG1's specific characteristics and the requirements of downstream applications.

How can recombinant PSG1 be utilized to identify its receptor in human cells?

Identifying the human PSG1 receptor presents a significant challenge, as it doesn't utilize CD9 like mouse PSG17 . Several methodological approaches can be employed:

• Protein Interaction Screening:

  • Affinity purification using immobilized PSG1 followed by mass spectrometry

  • Yeast two-hybrid screening with PSG1 as bait against human macrophage cDNA libraries

  • Proximity labeling approaches (BioID, APEX) with PSG1 fusion proteins

  • Protein microarray screening with labeled PSG1

• Cross-linking Studies:

  • Chemical cross-linking of PSG1 to its binding partners on human macrophages

  • Photo-cross-linking using PSG1 with incorporated photo-activatable amino acids

  • Analysis of cross-linked complexes by mass spectrometry

  • Validation of candidates through targeted knockdown or knockout approaches

• Cell-based Binding Assays:

  • Flow cytometry with fluorescently-labeled PSG1 on various human cell types

  • Surface plasmon resonance with recombinant receptor candidates

  • Competition assays with truncated PSG1 domains to map binding regions

  • Systematic screening of human membrane protein libraries

• Comparative Genomics Approaches:

  • Analysis of gene expression correlation between PSG1 and potential receptors

  • Evolutionary analysis to identify proteins under co-evolutionary pressure with PSGs

  • Computational prediction of protein-protein interactions

• Functional Validation:

  • CRISPR-Cas9 knockout of candidate receptors followed by PSG1 response testing

  • Reconstitution experiments in receptor-negative cell lines

  • Structure-function studies of PSG1-receptor interactions using mutational analysis

These complementary approaches can overcome the challenges in identifying the elusive human PSG1 receptor, providing critical insights into its signaling mechanisms and potentially revealing new therapeutic targets for pregnancy-related disorders.

What role might PSG1 play in pregnancy complications, and how can Sf9-expressed PSG1 help investigate this?

PSG1's immunomodulatory functions suggest potential roles in pregnancy complications, which can be investigated using recombinant PSG1 from Sf9 cells:

• Expression Level Studies:

  • Compare PSG1 levels in normal versus complicated pregnancies

  • Develop standardized ELISAs using recombinant PSG1 as calibration standards

  • Investigate correlation between PSG1 levels and pregnancy outcomes

• Functional Variation Analysis:

  • Express naturally occurring PSG1 variants in Sf9 cells

  • Compare their activity in cytokine induction assays

  • Investigate whether specific variants correlate with pregnancy complications

• Immune Dysfunction Models:

  • Use recombinant PSG1 to restore immune balance in ex vivo models of pregnancy complications

  • Investigate PSG1's effects on specific immune cell populations implicated in conditions like preeclampsia or recurrent miscarriage

  • Determine whether PSG1 can correct TH1/TH2 imbalances associated with pregnancy loss

• Placental Dysfunction Studies:

  • Examine PSG1's effects on trophoblast invasion and spiral artery remodeling

  • Investigate relationships between PSG1 and other placental factors implicated in complications

  • Develop in vitro models using primary trophoblasts and recombinant PSG1

• Biomarker Development:

  • Use recombinant PSG1 to develop and validate antibodies for diagnostic applications

  • Create multiplexed assays measuring PSG1 alongside other pregnancy biomarkers

  • Assess PSG1's utility as a predictive biomarker for complications

• Therapeutic Potential Exploration:

  • Test recombinant PSG1 as an immunomodulatory agent in animal models of pregnancy complications

  • Investigate dose-response relationships and administration routes

  • Explore PSG1-derived peptides with enhanced stability or specificity

The use of well-characterized, recombinant PSG1 from Sf9 cells enables standardized, reproducible experiments critical for understanding its role in pregnancy complications and exploring potential therapeutic applications.

How can comparative studies between human PSG1 and mouse PSGs contribute to evolutionary understanding of placentation?

The search results highlight significant differences between human and mouse PSGs despite similar functions , offering unique opportunities for evolutionary insights:

• Receptor Divergence Analysis:

  • The search results clearly demonstrate that mouse PSG17 binds CD9, while human PSGs use a different, unidentified receptor

  • Compare receptor binding domains across species to track evolutionary changes

  • Investigate whether receptor switching occurred during evolution or if different receptors were recruited independently

• Functional Conservation Testing:

  • Despite different receptors, both human and mouse PSGs induce similar anti-inflammatory cytokines

  • Use recombinant proteins to compare cytokine induction profiles across species

  • Determine whether the same downstream signaling pathways are activated despite different initial receptor interactions

• Structural Comparative Analysis:

  • Express and purify both human PSG1 and mouse PSG17 in Sf9 cells using similar protocols

  • Compare their structural features through various biophysical techniques

  • Identify conserved structural elements that might be critical for function despite sequence divergence

• Domain Swapping Experiments:

  • Create chimeric proteins between human PSG1 and mouse PSG17

  • Express these in Sf9 cells and test their activity and receptor preference

  • Map the evolutionary changes in functional domains

• Phylogenetic Studies:

  • Expand comparative analysis to PSGs from other species with hemochorial placentation

  • Correlate PSG structural/functional features with placentation depth and invasiveness

  • Test the hypothesis mentioned in the search results that PSGs have "an essential role in the regulation of the maternal immune response in species with hemochorial placentation"

• Selective Pressure Analysis:

  • Identify regions of PSG genes under positive or purifying selection

  • Correlate these with functional domains and receptor interactions

  • Investigate whether immune evasion has driven PSG evolution

These comparative approaches can provide valuable insights into how different molecular mechanisms evolved to fulfill similar immunomodulatory functions critical for successful pregnancy across mammalian species.