Recombinant Saguinus labiatus Alpha-synuclein (SNCA)
Description
Introduction
Alpha-synuclein (SNCA) is a 140-amino-acid protein primarily expressed in the brain, known for its role in neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). While SNCA has been extensively studied in humans and model organisms like mice, recombinant SNCA from Saguinus labiatus (a species of tamarin monkey) remains unexplored in the provided search results. This article synthesizes available data on SNCA biology and production methods, while noting the absence of specific studies on Saguinus labiatus SNCA.
Background and Structure
Alpha-synuclein exists in three structural regions:
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Amino terminus (residues 1–60): Contains lipid-binding motifs that form α-helical structures upon membrane binding .
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Central hydrophobic region (61–95): The NAC domain, critical for β-sheet formation and aggregation .
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Carboxyl terminus (96–140): Unstructured and negatively charged, prone to post-translational modifications .
| Property | Value |
|---|---|
| Molecular weight | ~14.4 kDa (theoretical) |
| Expression system | E. coli or HEK293 |
| Purity | >95% (SDS-PAGE) |
Production Methods
Recombinant SNCA is typically produced via bacterial (E. coli) or mammalian (HEK293) systems. Key steps include:
Research Applications
SNCA research focuses on:
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Neurodegenerative diseases: Fibrillar SNCA aggregates are central to Lewy bodies in PD and amyloid plaques in AD .
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Toxicity mechanisms: Oligomers (e.g., SPR-484) induce neuronal damage and serine 129 phosphorylation .
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Therapeutic targets: Inhibitors of aggregation or clearance enhancers .
| Disease | SNCA Pathology |
|---|---|
| Parkinson’s disease | Lewy body inclusions |
| Alzheimer’s disease | Amyloid plaques |
| Multiple system atrophy | Glial cytoplasmic inclusions |
Challenges in Saguinus labiatus Studies
No data on Saguinus labiatus SNCA exist in the provided sources. Key challenges for studying this species include:
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Limited species-specific tools: Antibodies and expression systems optimized for human/mouse SNCA .
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Phylogenetic divergence: Sequence differences may alter aggregation or toxicity .
Future Directions
Product Specs
Target Background
Q&A
Basic Research Questions
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What is the difference between human alpha-synuclein and Saguinus labiatus (white-lipped tamarin) alpha-synuclein protein sequences?
New World monkeys, including Saguinus labiatus (white-lipped tamarin), exhibit significant differences in their alpha-synuclein protein sequences compared to humans. While Great Apes (gorillas, orangutans, and bonobos) possess identical protein sequences to humans, white-lipped tamarins have alterations at amino acid positions 53, 87, 99, and 103. These differences are particularly noteworthy as position 53 corresponds to the A53T mutation site in humans, which is associated with early-onset familial Parkinson's disease . This natural variation makes S. labiatus alpha-synuclein valuable for comparative studies of protein aggregation and pathogenicity.
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What expression systems are most appropriate for producing recombinant alpha-synuclein from New World monkeys?
E. coli remains the preferred expression system for recombinant alpha-synuclein from all species, including New World monkeys. The BL21(DE3) strain is particularly effective for high-density cell culture-based expression systems. When expressing S. labiatus alpha-synuclein, consider these methodological approaches:
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Use of pET expression vectors with IPTG induction (0.5-1.0 mM)
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Induction at OD600 of 0.6-0.8
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Expression at lower temperatures (25-30°C for 4-6 hours) to enhance solubility
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Supplementation with glucose (0.5-1%) to prevent leaky expression before induction
Recent advances in cell-engineered recombinant alpha-synuclein production have demonstrated improved batch-to-batch consistency when validated through gage reproducibility and repeatability (Gage R&R) methodologies .
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What purification methods yield the highest purity for recombinant alpha-synuclein from New World monkeys?
Four primary extraction methods have been documented for alpha-synuclein purification, with varying impacts on protein properties:
Extraction Method Advantages Disadvantages Impact on Aggregation Propensity Boiling (95-100°C) Removes most heat-sensitive bacterial proteins Possible partial degradation with extended boiling Moderate Acid precipitation High purity Potential for protein modification Lower Ammonium sulfate precipitation Good yield Additional purification steps required Higher Periplasmic lysis Reduced endotoxin Complex procedure Higher For S. labiatus alpha-synuclein specifically, a two-step chromatography approach is recommended: anion exchange chromatography followed by size exclusion chromatography, as this eliminates both bacterial contaminants and potential oligomeric species that could seed aggregation .
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How do I verify the quality and structural properties of recombinant S. labiatus alpha-synuclein?
Quality assessment should employ multiple orthogonal techniques:
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SDS-PAGE for purity assessment (>95% purity standard)
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Western blotting using SNCA-specific antibodies
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Mass spectrometry to confirm molecular weight and sequence
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Circular dichroism to assess secondary structure (should show predominantly unfolded state)
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Dynamic light scattering to confirm monomeric state
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ThT fluorescence assay to confirm absence of pre-formed aggregates
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Endotoxin testing (<1.0 EU per μg protein is considered acceptable)
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Experimental Design Considerations
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What controls should be included when studying aggregation properties of S. labiatus alpha-synuclein compared to human alpha-synuclein?
Rigorous experimental design requires appropriate controls:
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Monomeric protein controls: Both freshly prepared S. labiatus and human alpha-synuclein
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Buffer-only controls to detect contamination
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Cross-seeding controls: Test whether pre-formed fibrils of each species can seed the other
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A53T human mutant as a reference point for S. labiatus behavior
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Known aggregation modifiers (e.g., dopamine, heparin) to validate normal response patterns
All experiments should be performed with proteins prepared using identical protocols to eliminate methodology-based variations. Multivariate analysis approaches, particularly principal component analysis (PCA), have proven valuable for identifying subtle differences in aggregation behavior between species variants .
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How can I effectively use S. labiatus alpha-synuclein to study the prion-like propagation hypothesis in Parkinson's disease?
The natural "A53T-like" sequence of S. labiatus alpha-synuclein provides unique opportunities for studying prion-like propagation:
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Preparation of fluorescently labeled monomers (using Alexa Fluor dyes or equivalent)
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Generation of stable seed preparations:
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Sonicated pre-formed fibrils (PFFs)
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Brain-derived seeds from transgenic models
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Exosome-associated alpha-synuclein
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Experimental readouts:
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Cell-to-cell transfer using microfluidic chambers
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Templated aggregation using ThT or alpha-synuclein-GFP reporter cells
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Phosphorylation at S129 using immunohistochemistry
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Neuronal dysfunction through calcium imaging or electrophysiology
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Research has demonstrated that alpha-synuclein can be transferred from gut mucosal cells to the vagus nerve and ultimately to the hindbrain, providing an important model system for studying prion-like propagation mechanisms in Parkinson's disease pathogenesis .
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