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E3 Ligase or Target Proteins Expression and Purification
E3 Ligase or Target Proteins Expression and Purification
At Profacgen, our E3 Ligase and Target Protein Expression and Purification Service delivers high-quality recombinant proteins from diverse expression systems, supporting ligand screening, ternary complex studies, ubiquitination assays, and structural biology for targeted protein degradation programs.
E3 ubiquitin ligases fall into three major classes: RING, HECT, and RBR. RING E3s feature zinc-binding RING or U-box domains and include multi-subunit complexes such as cullin-RING ligases (CRLs) and APC/C. HECT E3s adopt a bilobal architecture with flexible N- and C-terminal lobes, divided into Nedd4, HERC, and other subfamilies. RBR E3s contain RING1 and RING2 domains separated by an IBR domain. Many E3 ligases possess extensive unstructured or random coil regions, posing challenges for expression and purification. Profacgen provides comprehensive protein expression services across multiple host systems to support E3 ligase and target protein production for functional assays, target validation, and structural studies.
Overview
High-quality protein production is foundational to every stage of targeted protein degradation research. Our platform addresses the diverse structural and physicochemical challenges of E3 ligases and target proteins:
E3 ligases: Full-length and domain constructs of RING, HECT, and RBR family members, including multi-subunit complexes requiring co-expression of regulatory subunits and post-translational modification enzymes
Target proteins: Disease-relevant substrates including kinases, transcription factors, epigenetic regulators, and scaffold proteins with varying solubility, stability, and folding requirements
Protein domains: Isolated functional domains, substrate-binding regions, and catalytic cores that enable focused structural and interaction studies while circumventing expression challenges of full-length proteins
Mutants: Site-directed mutants, deletion constructs, and disease-associated variants for mechanistic studies, SAR analysis, and validation of structure-function relationships
Service Workflow
Expression Platforms
Profacgen employs multiple expression systems matched to protein complexity, modification requirements, and downstream applications:
Bacterial Expression
Cost-effective production of soluble domains and proteins requiring no post-translational modifications.
Escherichia coli: Rapid, high-yield expression of cytoplasmic and periplasmic proteins with diverse vector and strain options
Solubility enhancement: Co-expression of chaperones, fusion tags, and low-temperature induction strategies to improve folding
Isotopic labeling: 15N, 13C, and 2H labeling for NMR structural studies
Mammalian Expression
Native folding and post-translational modification for complex E3 ligases and target proteins.
HEK293 and CHO systems: Transient and stable expression with serum-free and chemically defined media options
Post-translational modifications: Glycosylation, phosphorylation, and ubiquitination for physiologically relevant protein states
Secreted proteins: Signal peptide optimization and medium exchange for efficient recovery of extracellular domains
Insect Cell Expression
Balanced yield and modification capability for challenging multi-domain proteins.
Baculovirus system: High-titer viral stocks and optimized infection protocols for consistent protein production
Complex assemblies: Co-expression of multiple subunits for CRL complexes, APC/C, and other multi-component E3 ligases
Membrane proteins: E3 ligase-associated transmembrane receptors and ubiquitin receptors for interaction studies
Cell-Free Expression
Rapid production and specialized applications without cellular toxicity constraints.
Wheat germ and E. coli lysates: Open systems enabling incorporation of non-natural amino acids, labeled compounds, and redox control
Toxic proteins: Expression of proteins detrimental to cell viability, including proteases and membrane-disrupting domains
High-throughput: Parallel screening of expression conditions and construct variants in microplate formats
Purification Capabilities
Profacgen implements multi-step purification strategies tailored to protein properties and purity requirements:
Affinity purification: Immobilized metal affinity chromatography (IMAC) for His-tagged proteins, glutathione affinity for GST fusions, and streptavidin capture for biotinylated constructs. Tag removal by TEV, HRV 3C, or thrombin proteases available upon request
Ion exchange chromatography: Anion and cation exchange separation based on surface charge distribution, enabling resolution of charge variants, truncation products, and post-translational modification isoforms
Size exclusion chromatography: Gel filtration for aggregate removal, buffer exchange, and assessment of oligomeric state, hydrodynamic radius, and complex stoichiometry
Quality Characterization
Comprehensive analytical validation ensures protein suitability for downstream applications:
Purity analysis: SDS-PAGE and analytical SEC for visual and quantitative purity assessment, with threshold reporting to match application requirements
Activity verification: Functional assays including ubiquitination activity for E3 ligases, kinase activity for targets, and binding assays for interaction domains
Stability assessment: Thermal shift analysis, differential scanning fluorimetry, and long-term storage stability monitoring to guide formulation and handling protocols
Structural integrity evaluation: Circular dichroism for secondary structure content, dynamic light scattering for polydispersity, and analytical ultracentrifugation for sedimentation behavior
Supported Protein Types
Profacgen has established expression and purification protocols for major degrader-relevant protein classes:
CRBN: Full-length and substrate-binding domain constructs from multiple species, including disease-relevant mutants and post-translational modification variants
VHL: Elongin B/C complex components, VHL variants with altered substrate specificity, and hypoxia-inducible factor interaction domains
MDM2: p53-binding domain, RING domain, and full-length constructs with co-expression of MDMX for heterodimeric complex formation
cIAP: BIR domains, RING domains, and full-length proteins with co-expression partners for activation state modulation
Disease-relevant targets: Oncogenic kinases, transcription factors, epigenetic regulators, and neurodegeneration-associated proteins with customized solubility and stability engineering
Applications
Our expressed and purified proteins support diverse targeted protein degradation applications:
Ligand screening: High-quality protein reagents for biophysical binding assays, fragment screening, and virtual screening validation
Ternary complex studies: Purified E3 ligases, target proteins, and degrader components for in vitro reconstitution of ternary complexes and cooperativity analysis
Ubiquitination assays: Catalytically competent E3 ligases with matched E2 conjugating enzymes and substrates for activity profiling and inhibitor screening
Structural biology: Crystallization-grade proteins for X-ray crystallography, cryo-EM, and NMR spectroscopy of degrader-relevant complexes
Customize Protein Synthesis: Tailored expression constructs, host systems, and purification strategies matched to each protein's structural complexity and application requirements.
High Production and Recovery Rate: Optimized protocols, solubility engineering, and multi-system screening maximize yield and minimize loss during purification.
Scale-Up Protein Production: Seamless progression from milligram-scale discovery to gram-scale supply for screening, structural studies, and assay development.
Timely Feedback on Project Progress: Regular milestone updates, transparent communication, and proactive troubleshooting to maintain program timelines.
Representative Program Scenarios
Scenario 1: Multi-Subunit CRL Complex Expression
Program Context:
A degrader program required a cullin-RING ligase (CRL) complex comprising cullin, RING protein, substrate adapter, and target protein for ternary complex reconstitution. Individual subunits expressed poorly and failed to assemble functionally.
Objective:
To produce a fully assembled, catalytically active CRL complex with correct subunit stoichiometry and ubiquitination activity suitable for degrader screening.
Approach:
Profacgen employed a baculovirus co-infection strategy in insect cells, optimizing the ratio of viral stocks for each subunit to achieve balanced expression. A polyprotein construct with self-cleaving linkers was tested as an alternative approach. Complex assembly was monitored by co-immunoprecipitation and analytical SEC. Active complexes were purified by sequential affinity and size exclusion chromatography, then validated by in vitro ubiquitination assays with model substrates.
Outcome:
The optimized co-infection protocol yielded milligram quantities of assembled CRL complex with correct 1:1:1:1 stoichiometry. The complex demonstrated robust E3 ligase activity and supported ternary complex formation with a candidate PROTAC, enabling quantitative SPR analysis and cellular degradation validation.
Scenario 2: Disease-Associated Target Kinase with Low Solubility
Program Context:
An oncology target kinase was required for fragment screening and degrader warhead optimization. Previous expression attempts in bacterial systems yielded insoluble aggregate with no detectable kinase activity.
Objective:
To produce soluble, active kinase with confirmed ligand binding capability and structural integrity suitable for biophysical screening and crystallography.
Approach:
Profacgen screened multiple expression systems (bacterial with chaperone co-expression, insect cell, and mammalian) and evaluated diverse N-terminal fusion tags (Trx, MBP, NusA) and surface entropy reduction mutations. Solubility and activity were assessed by analytical SEC and kinase assay. The optimal construct was produced in mammalian cells with a cleavable MBP tag, yielding soluble protein after tag removal.
Outcome:
The optimized kinase was delivered at >95% purity with confirmed ATPase activity and sub-micromolar inhibitor binding. The protein supported successful fragment screening by X-ray crystallography, yielding multiple co-crystal structures that guided degrader warhead design and linker placement.
Q: Which expression system is best for my protein?
A: Selection depends on protein complexity and application. Bacterial systems are cost-effective for simple cytoplasmic proteins without modifications. Mammalian and insect cells are preferred for multi-domain E3 ligases, glycoproteins, and complexes requiring assembly. We screen multiple systems in parallel when the optimal approach is uncertain.
Q: Can you express multi-subunit E3 ligase complexes?
A: Yes. We routinely produce CRL complexes, APC/C subassemblies, and heterodimeric RING E3s by co-infection or co-transfection strategies. Polyprotein constructs with self-cleaving linkers are employed when co-expression is inefficient. Complex assembly is verified by analytical SEC and functional assays.
Q: What purity levels can you achieve?
A: We routinely achieve >95% purity for most proteins, with >98% available for structural biology applications. Purity is assessed by densitometric SDS-PAGE and analytical SEC. Custom purity specifications are accommodated based on downstream requirements.
Q: Do you provide endotoxin-free proteins?
A: Yes. Endotoxin removal is standard for mammalian and insect cell-derived proteins. For bacterial proteins, we offer additional endotoxin reduction steps including polymyxin B affinity chromatography and Triton X-114 phase separation. Endotoxin levels are quantified by LAL assay with reporting to <0.1 EU/µg upon request.
Q: Can you produce isotopically labeled proteins for NMR?
A: Yes. We offer 15N, 13C, and 2H labeling in bacterial and cell-free expression systems. Selenomethionine incorporation for phasing in X-ray crystallography is also available. Labeling efficiency is verified by mass spectrometry.
Q: What is the typical timeline from gene to purified protein?
A: Standard bacterial expression delivers purified protein in 3–4 weeks. Mammalian and insect cell systems require 5–8 weeks due to longer cell culture and viral amplification steps. Complex multi-subunit proteins or challenging targets may extend timelines by 2–4 weeks for optimization. Rush services are available for urgent projects.
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