Recombinant Bovine Spastin (SPAST)
Description
Introduction to Recombinant Bovine Spastin (SPAST)
Spastin, encoded by the SPAST gene, is a highly conserved ATPase protein found in organisms ranging from insects to mammals . It belongs to the AAA (ATPases Associated with diverse cellular Activities) protein family and participates in various cellular processes, including membrane trafficking, intracellular motility, organelle biogenesis, protein folding, and proteolysis . Recombinant Bovine Spastin is produced using genetic engineering techniques to generate the bovine (cow) version of the spastin protein in a laboratory setting, often for research purposes .
Gene and Protein Structure
The SPAST gene codes for spastin, a microtubule-severing protein . Human spastin has two transcript variants encoding distinct isoforms . Alternative splicing can result in multiple transcript variants that encode isoforms that differ in protein regions . Spastin monomers assemble into hexameric, ring-shaped ATPases that sever microtubules along their lengths . Each spastin monomer contains a carboxyl-terminal AAA ATPase catalytic domain and a distinct microtubule-interacting and trafficking (MIT) domain .
Function and Mechanism of Action
Spastin severs microtubules along their lengths, a process distinct from dynamic instability, which occurs spontaneously only at microtubule ends . The AAA domain is critical for its function; most spastin mutations identified in AD-HSP patient cohorts compromise its ATPase activity .
Role in Disease
Mutations in the SPAST gene are the most frequent cause of autosomal dominant hereditary spastic paraplegia (AD-HSP) . In cattle, a mutation in the SPAST gene is associated with congenital bovine spinal dysmyelination (BSD) .
Recombinant Spastin Production and Research Applications
Recombinant spastin proteins are created for in vitro studies to examine the effects of mutations on ATPase activity and microtubule severing . For example, recombinant fusion proteins between spastin and GST (Glutathione S-transferase) have been expressed in E. coli and purified by affinity chromatography to measure ATPase activity . Studies using recombinant spastin have demonstrated that mutations, such as the R560Q substitution found in BSD, can abolish ATPase activity, establishing a causal relationship between loss of catalytic activity and the disease .
Impact of Mutations on Spastin Function
Spastin Isoforms and Their Functional Differences
Alternative splicing of the SPAST gene results in different isoforms with varying N-terminal lengths, which affects their efficiency in nuclear export . M1 and M87 are two such isoforms; studies have shown that mutant M1 spastin proteins can have toxic effects on axonal transport, independent of microtubule-binding and severing activities .
Product Specs
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Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C. Lyophilized formulations have a 12-month shelf life at -20°C/-80°C.
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Target Background
- Studies have established a direct link between SPAST mutations and bovine spinal dysmyelination. PMID: 19714378
- A defective SPAST gene in cattle is associated with a recessive, congenital neurodegenerative disorder characterized by pathological myelin sheath alterations in the spinal cord. PMID: 19714378
Q&A
What is bovine SPAST and what is its primary function?
Bovine SPAST encodes a microtubule-severing protein called spastin that plays a critical role in neuronal function. The primary function of spastin is to sever microtubules into smaller segments, which facilitates microtubule reorganization crucial for neurite growth and regeneration . This protein belongs to the AAA (ATPases Associated with diverse cellular Activities) family, which requires ATP hydrolysis to perform its severing function. In cattle, SPAST is particularly important for proper myelination in the spinal cord, and mutations in this gene have been linked to bovine spinal dysmyelination (BSD) .
What are the different isoforms of bovine SPAST?
Similar to human SPAST, bovine SPAST expresses multiple isoforms, with the two main variants being analogous to human M1 (full-length) and M87 (shorter) isoforms. The M1 isoform contains an N-terminal region that is absent in M87. This distinction is functionally significant as:
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The M1 isoform shows tissue-specific expression predominantly in the adult spinal cord
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The M87 isoform is ubiquitously expressed across various tissues
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Only the M1 isoform with mutations has been shown to inhibit fast axonal transport (FAT) in experimental models
Understanding these isoform differences is crucial when designing experiments with recombinant bovine SPAST, as the isoform selection can significantly impact observed biological effects.
How does the ATPase domain function in bovine SPAST?
The ATPase domain in bovine SPAST is highly conserved and critical for its microtubule-severing activity. This domain:
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Binds and hydrolyzes ATP to generate energy for microtubule severing
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Contains sites where disease-causing mutations frequently occur
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Includes the R560 residue, which when mutated to glutamine (R560Q) in cattle, severely impairs ATPase activity and causes BSD
Experimental approaches to study this domain typically involve site-directed mutagenesis to create recombinant proteins with specific amino acid substitutions, followed by in vitro ATPase activity assays to measure functional consequences.
What expression systems yield functional recombinant bovine SPAST?
When expressing recombinant bovine SPAST, the choice of expression system is critical for obtaining properly folded, active protein. Based on published methodologies:
| Expression System | Advantages | Limitations | Best For |
|---|---|---|---|
| E. coli | High yield, cost-effective, rapid production | May lack post-translational modifications, inclusion body formation common | Structure studies, antibody production |
| Insect cells | Better folding, some post-translational modifications | More complex, moderate yield | Functional assays, protein-protein interaction studies |
| Mammalian cells | Native-like folding and modifications | Lower yield, higher cost, time-consuming | Cellular localization studies, in vivo activity assays |
For functional studies of bovine SPAST's microtubule-severing activity, insect or mammalian cell expression systems are generally preferred to ensure proper protein folding and post-translational modifications that may be crucial for enzyme activity.
What purification methods yield the highest activity of recombinant bovine SPAST?
A methodological approach for high-yield purification of active recombinant bovine SPAST typically follows these steps:
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Generate expression constructs with appropriate tags (His, GST, or FLAG) for the selected isoform (M1 or M87)
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Express protein under optimized conditions (temperature, induction time, media composition)
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Lyse cells using mild detergents to preserve protein structure
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Implement a two-step purification process:
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Initial affinity chromatography based on the fusion tag
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Secondary size-exclusion or ion-exchange chromatography for higher purity
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Verify purification by SDS-PAGE and Western blotting with anti-SPAST antibodies
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Perform activity assays to confirm functional integrity
For bovine SPAST specifically, maintaining the protein in buffers containing ATP or non-hydrolyzable ATP analogs during purification can help stabilize the protein structure and preserve its activity.
How can I measure the microtubule-severing activity of recombinant bovine SPAST?
To quantitatively assess microtubule-severing activity of recombinant bovine SPAST, researchers can employ several complementary methodologies:
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In vitro microtubule severing assay:
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Polymerize fluorescently labeled tubulin into microtubules
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Immobilize microtubules on glass coverslips
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Add purified recombinant SPAST at defined concentrations
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Use time-lapse microscopy to visualize and quantify severing events
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Calculate severing rate as the number of breaks per microtubule length per time
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ATPase activity coupling:
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Measure ATP hydrolysis rate using a coupled enzymatic assay
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Compare ATPase activity in the presence and absence of microtubules
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Calculate the stimulation of ATPase activity by microtubules as an indirect measure of binding and function
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Cellular microtubule network analysis:
These approaches should be used in combination to comprehensively characterize SPAST activity.
How does ATP concentration affect the activity of recombinant bovine SPAST?
The ATPase activity of bovine SPAST follows Michaelis-Menten kinetics, with important experimental considerations:
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At low ATP concentrations (<100 μM), activity increases linearly with ATP concentration
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The Km for ATP typically falls in the range of 200-400 μM for wild-type SPAST
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Disease-causing mutations like R560Q show significantly impaired ATPase activity with much higher Km values
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ATP concentrations ≥2 mM are typically used for in vitro severing assays to ensure maximum activity
When conducting kinetic experiments, researchers should:
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Use a range of ATP concentrations (50 μM to 2 mM)
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Include appropriate controls (no ATP, non-hydrolyzable ATP analogs)
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Compare wild-type and mutant proteins under identical conditions
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Account for the effects of divalent cations (Mg2+) which are required cofactors
How does the R560Q mutation affect bovine SPAST function?
The R560Q mutation in bovine SPAST represents a critical model for understanding structure-function relationships. Experimental evidence demonstrates that:
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The R560 residue is located in the highly conserved ATPase domain
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This position is invariant from insects to mammals, indicating its evolutionary importance
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The R560Q substitution severely impairs ATPase activity in biochemical assays
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Homozygous R560Q mutations cause bovine spinal dysmyelination (BSD), a recessive congenital neurodegenerative disease
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Equivalent mutations in human SPAST cause hereditary spastic paraplegia (HSP)
Research methods to analyze this mutation include:
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Recombinant protein expression of wild-type and R560Q mutant versions
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Comparative ATPase activity assays
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Microtubule binding and severing assays
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Structural studies to understand the molecular basis for dysfunction
These approaches have demonstrated that the R560Q mutation significantly reduces the ATPase activity of bovine SPAST, providing a direct causal link between this molecular defect and the observed disease phenotype .
How can cellular models be used to study bovine SPAST mutations?
Cellular models provide valuable systems for investigating the pathophysiological mechanisms of bovine SPAST mutations. Effective methodological approaches include:
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Primary neuronal cultures:
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Isolate neurons from bovine embryos with different SPAST genotypes
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Analyze neurite outgrowth, branching, and regeneration after injury
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Evaluate microtubule dynamics and organization in real-time
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Transfected cell lines:
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Express wild-type or mutant bovine SPAST in neuronal or non-neuronal cell lines
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Assess effects on microtubule network, organelle distribution, and cellular morphology
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Perform live-cell imaging to track cellular dynamics
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CRISPR/Cas9 gene editing:
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Generate isogenic cellular models with specific SPAST mutations
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Study mutation-specific effects in a controlled genetic background
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Compare different mutations to establish genotype-phenotype correlations
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These cellular models can reveal functional consequences of SPAST mutations, particularly how they affect axonal transport, a process critical for neuronal health and implicated in neurodegenerative diseases associated with SPAST mutations .
How does the 14-3-3/spastin pathway regulate neuronal regeneration?
The interaction between 14-3-3 proteins and spastin represents an important regulatory mechanism with implications for neuronal regeneration. Research methodologies to study this pathway include:
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Co-immunoprecipitation assays:
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Phosphorylation site analysis:
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Protein stability assays:
After spinal cord injury, spastin levels initially decrease but gradually recover over a 30-day period. Experimental enhancement of the 14-3-3/spastin interaction using compounds like FC-A can increase spastin levels and potentially promote neuronal regeneration following injury .
What role does bovine SPAST play in axonal transport?
Research on spastin's impact on fast axonal transport (FAT) has revealed isoform-specific effects relevant to disease pathology. Key findings and methodologies include:
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Isolated axoplasm studies:
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Live-cell transport assays:
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Tracking labeled vesicles, mitochondria, or other organelles in cultured neurons
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Measuring transport velocities, run lengths, and pause frequencies
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Comparing effects of wild-type versus mutant bovine SPAST expression
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Mechanistic investigations:
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Casein kinase 2 (CK2) has been identified as a critical mediator of mutant spastin-induced FAT deficits
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Pharmacological CK2 inhibitors can correct organelle distribution abnormalities caused by mutant M1 spastin
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Similar approaches can determine if bovine SPAST mutations operate through the same mechanisms
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These methodologies have established that pathogenic effects of mutant spastin on axonal transport are specific to the M1 isoform and independent of microtubule-binding or severing activity, highlighting the importance of studying both major isoforms in bovine models .
How should controls be designed for bovine SPAST functional studies?
Proper experimental controls are essential for reliable interpretation of bovine SPAST functional studies. A comprehensive control strategy should include:
Adhering to these control strategies ensures experimental rigor and facilitates cross-study comparisons in the scientific literature.
How can I resolve contradictory data when studying bovine SPAST?
When faced with contradictory results in bovine SPAST research, consider the following methodological approaches:
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Isoform verification:
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Expression level considerations:
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Post-translational modification status:
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Experimental system differences:
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In vitro vs. cellular vs. in vivo systems can yield different results
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Species-specific differences in SPAST regulation may exist
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Cell-type specific effects need to be considered (neuronal vs. non-neuronal cells)
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By systematically addressing these variables, researchers can often reconcile seemingly contradictory findings and develop a more comprehensive understanding of bovine SPAST biology.
