SERPINC1 Human, Sf9
Description
Molecular Characterization
SERPINC1 Human, Sf9 is synthesized as a single glycosylated polypeptide chain containing 441 amino acids (residues 33–464) with a molecular mass of 50.1 kDa. Post-translational modifications in Sf9 cells result in an apparent molecular weight of 50–70 kDa on SDS-PAGE due to glycosylation . The protein includes a C-terminal 9-amino-acid histidine tag for purification and retains critical functional domains:
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Heparin-binding domain (N-terminal)
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Reactive site loop (C-terminal) with Arg393-Ser394 scissile bond
Key physical properties:
Biological Function
SERPINC1 Human, Sf9 exhibits potent anticoagulant activity by:
Deficiencies in SERPINC1 are linked to hereditary thrombophilia, with >220 mutations identified (e.g., type I: reduced synthesis; type II: functional defects) .
Production and Purification
The recombinant protein is generated through:
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Chromatographic purification using nickel affinity (His-tag) and proprietary methods
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Quality control via SDS-PAGE and functional assays (e.g., heparin-binding capacity)
Amino Acid Sequence Highlights:
4.1. Disease Modeling
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Used to study antithrombin deficiency mechanisms, including mutations like Cambridge II (Ala384Ser) .
4.2. Therapeutic Development
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Preclinical studies demonstrate efficacy in reducing arterial plaque formation and inflammation at picogram doses .
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PEGylated SERPINC1 derivatives show promise in treating viral infections (e.g., SARS-CoV-2) by modulating immune responses .
Comparative Advantages of Sf9 Expression
Product Specs
SERPINC1, AT3, AT3D, ATIII, THPH7, Antithrombin-III, Serpin C1.
Sf9, Baculovirus cells.
ADPHGSPVDI CTAKPRDIPM NPMCIYRSPE KKATEDEGSE QKIPEATNRR VWELSKANSR FATTFYQHLA DSKNDNDNIF LSPLSISTAF AMTKLGACND TLQQLMEVFK FDTISEKTSD QIHFFFAKLN CRLYRKANKS SKLVSANRLF GDKSLTFNET YQDISELVYG AKLQPLDFKE NAEQSRAAIN KWVSNKTEGR ITDVIPSEAI NELTVLVLVN TIYFKGLWKS KFSPENTRKE LFYKADGESC SASMMYQEGK FRYRRVAEGT QVLELPFKGD DITMVLILPK PEKSLAKVEK ELTPEVLQEW LDELEEMMLV VHMPRFRIED GFSLKEQLQD MGLVDLFSPE KSKLPGIVAE GRDDLYVSDA FHKAFLEVNE EGSEAAASTA VVIAGRSLNP NRVTFKANRP FLVFIREVPL NTIIFMGRVA NPCVKHHHHH H.
Q&A
What expression system considerations are critical for producing functional SERPINC1 in Sf9 cells?
Sf9 cells utilize baculovirus vectors to achieve post-translational modifications closer to mammalian systems than bacterial alternatives. The recombinant SERPINC1 described in sources contains a C-terminal 9xHis tag for purification via immobilized metal affinity chromatography (IMAC). Key parameters include:
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Codon Optimization: Insect cells exhibit biased tRNA pools; codon optimization of the human SERPINC1 gene improves translation efficiency.
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Glycosylation Profile: Sf9 cells add high-mannose N-glycans at Asn-135 and Asn-155, altering electrophoretic mobility (50–70 kDa on SDS-PAGE versus calculated 50.1 kDa) . Confirm glycosylation using EndoH digestion followed by western blot.
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Protease Inhibition: Validate activity early by testing thrombin inhibition kinetics (see Question 3).
How do researchers verify the structural integrity of recombinant SERPINC1?
Structural validation requires a multi-modal approach:
| Method | Target Parameter | Acceptance Criteria |
|---|---|---|
| Circular Dichroism | Secondary structure (α-helix/β-sheet ratio) | Match to human plasma-derived SERPINC1 |
| Size-Exclusion Chromatography | Oligomeric state | Monomeric peak (retention time = 14–16 min) |
| Mass Spectrometry | Molecular weight (± 1 Da) | 50,100 Da (non-glycosylated backbone) |
Discrepancies in SEC elution profiles may indicate aggregation—add 10% glycerol to storage buffers to stabilize the protein .
Which functional assays are optimal for quantifying SERPINC1 activity?
Two complementary assays are recommended:
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Chromogenic Thrombin Inhibition Assay
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Principle: Measure residual thrombin activity after incubation with SERPINC1 using a para-nitroaniline (pNA) substrate.
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Protocol:
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Pre-incubate 100 nM SERPINC1 with 10 nM thrombin for 60 sec.
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Add 0.5 mM Chromozym TH and record ΔA405/min.
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Calculate % inhibition relative to thrombin-only controls.
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Factor Xa Inhibition ELISA
Critical Note: Source demonstrates that assay reagents (e.g., APTT activator) significantly impact coagulation factor measurements. Always validate against a reference standard (e.g., NIBSC 17/264).
How should researchers resolve discrepancies between SERPINC1 activity assays and genetic data?
Inherited SERPINC1 mutations (e.g., Arg79Cys, Ser148Pro) may cause discordant functional results:
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Case Example: A patient with 65% anti-factor Xa activity but normal antigen levels suggests a dysfunctional variant. Follow-up steps:
Source reports that 68.4% of patients with reduced antithrombin activity harbor pathogenic SERPINC1 variants, emphasizing the need for combined biochemical/genetic analyses.
What strategies optimize SERPINC1 expression yields in Sf9 systems without compromising function?
Multiplicity of Infection (MOI) Optimization:
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Test MOIs from 0.1 to 5; higher MOIs increase protein yield but accelerate cell lysis. For SERPINC1, MOI = 1.0 typically balances yield (≈2 mg/L) and viability .
Post-Translational Modification Engineering:
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Co-express mammalian β-1,4-galactosyltransferase in Sf9 cells to modify N-glycans, improving serum half-life in preclinical models.
Protease Knockout Sf9 Lines:
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Use CRISPR-Cas9 to delete endogenous proteases (e.g., cathepsin L) that degrade SERPINC1 during secretion.
How can researchers integrate SERPINC1 data with multi-omics datasets in thrombosis studies?
Leverage platforms like Olink Proteomics to measure SERPINC1 alongside 1,472 other plasma proteins. In COVID-19 cohorts, source identified SERPINA3 and CRP as co-regulated inflammatory markers. For thrombosis research:
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Network Analysis: Construct protein-protein interaction networks using STRING (https://string-db.org) to identify modules enriched for coagulation factors.
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Machine Learning: Train random forest models on SERPINC1 activity, genetic variants, and clinical outcomes (e.g., stroke recurrence) to predict thrombotic risk .
Why do SDS-PAGE results show higher apparent molecular weights for recombinant SERPINC1?
The observed 50–70 kDa band (vs. 50.1 kDa theoretical) arises from:
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N-Linked Glycosylation: Sf9 cells add ≈2–3 kDa of glycans per site.
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C-Terminal His Tag: The 9xHis tag contributes ≈1.2 kDa but may slow migration.
Resolution:
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Deglycosylate with PNGase F and re-run SDS-PAGE.
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Compare with human plasma SERPINC1 (control).
What are the implications of SERPINC1 glycosylation heterogeneity for functional studies?
Glycosylation impacts:
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Heparin Affinity: Non-sialylated glycans reduce heparin-binding kinetics by 30% .
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Thermal Stability: Glycosylated SERPINC1 retains 80% activity after 24 hr at 37°C vs. 50% for aglycosylated forms.
Protocol Adjustment:
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For structural studies, produce aglycosylated SERPINC1 in Sf9 cells treated with tunicamycin.
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For functional assays, use glycosylated protein to mimic physiological conditions.
Product Science Overview
Serpin Peptidase Inhibitor, Clade C Member 1, also known as SERPINC1 or antithrombin III, is a crucial protein in the human body. It belongs to the serpin (serine protease inhibitor) superfamily, which plays a significant role in regulating various physiological processes, including blood coagulation, inflammation, and immune responses .
SERPINC1 is a plasma protease inhibitor that primarily inhibits thrombin and other activated serine proteases involved in the coagulation cascade. This inhibition is essential for maintaining the balance between coagulation and anticoagulation, preventing excessive clot formation . The protein is composed of 455 amino acids and has a molecular mass of approximately 51.4 kDa .
The recombinant form of SERPINC1 is produced using the Sf9 insect cell expression system. This system is widely used for producing recombinant proteins due to its ability to perform post-translational modifications similar to those in mammalian cells. The recombinant SERPINC1 is typically purified using chromatographic techniques to ensure high purity and activity .
Deficiencies in SERPINC1 can lead to antithrombin III deficiency, an autosomal dominant disorder that increases the risk of thrombosis. This condition can result in recurrent deep vein thrombosis, pulmonary embolism, and other thrombotic events . Recombinant SERPINC1 is used therapeutically to manage and treat patients with antithrombin III deficiency, providing a crucial tool in preventing and controlling thrombotic disorders .
Recombinant SERPINC1 has several applications in research and medicine:
- Therapeutic Use: It is used to treat patients with antithrombin III deficiency, helping to prevent thrombotic events.
- Research: It serves as a valuable tool in studying the mechanisms of blood coagulation and the role of serpins in various physiological processes.
- Biotechnology: The recombinant protein is used in the development of anticoagulant therapies and other biotechnological applications .
