Recombinant Drosophila mojavensis Spastin (spas)
Description
Biochemical Properties
Recombinant Drosophila mojavensis Spastin is classified enzymatically as EC 3.6.4.3, identifying it as a hydrolase that acts on acid anhydrides to facilitate ATP-dependent microtubule severing. The protein is encoded by the spas gene and is referenced in protein databases under UniProt accession number B4K799 . Commercial preparations of this recombinant protein are typically produced in yeast expression systems and purified to >85% homogeneity as determined by SDS-PAGE analysis .
Microtubule Severing Mechanism
Spastin functions as a hexameric ring-shaped ATPase that severs microtubules along their lengths. This severing activity is distinct from the well-characterized disassembly mechanism of dynamic instability, which occurs spontaneously only at microtubule ends . The binding of spastin to microtubule polymers promotes its hexamerization and subsequent ATP hydrolysis, leading to the breakage of the microtubule substrate . This activity is crucial for regulating microtubule dynamics in neurons, particularly at synapses and within axons.
Neurodevelopmental Research Models
Drosophila has been successfully used as an in vivo model system to investigate spastin's functional role in regulating neuromuscular junction (NMJ) development and activity . Recombinant Drosophila mojavensis Spastin provides researchers with a valuable tool for comparative studies across Drosophila species, potentially revealing evolutionary adaptations in microtubule regulation.
Studies utilizing Drosophila models have demonstrated that spastin loss causes alterations in synaptic development of NMJs and defects in neurotransmission. These morphological and functional defects are associated with aberrant stabilization of the microtubule cytoskeleton at the pre-synapse . Different experimental approaches, including RNAi-mediated downregulation and expression of spastin missense mutations, have shown varying effects on NMJ development:
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RNAi-mediated reduction of spastin and expression of missense mutations lead to undergrowth of NMJ and specific reduction in synaptic area .
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Complete loss of spastin in null alleles results in increased bouton numbers and formation of abnormal grape-like clusters, linked to the absence of distal microtubules .
Human Disease Modeling
Mutations in human spastin (SPG4) represent the most common cause of autosomal dominant hereditary spastic paraplegia (AD-HSP), a neurodegenerative disorder characterized by progressive spasticity and weakness of the lower limbs . The functional conservation between Drosophila and human spastin makes Drosophila models, including those using recombinant Drosophila mojavensis Spastin, valuable for studying disease mechanisms.
Research has shown that specific mutations in the spastin AAA domain, such as the K388R substitution (equivalent to human K388R), cause complete loss of ATPase and microtubule severing activity in vitro . These findings have helped elucidate how disease-causing mutations might lead to pathology through either dominant-negative mechanisms or haploinsufficiency.
Therapeutic Potential
Recent research has identified potential therapeutic targets for spastin-related disorders. For instance, studies have shown that downregulation or chemical inhibition of CRL4, a regulator of spastin protein levels, can induce recovery of spastin levels and reduce HSP-associated phenotypes in Drosophila models of SPG4 haploinsufficiency and patient-derived cells . These findings suggest that manipulating spastin levels or activity could have therapeutic implications for human neurological disorders.
Expression and Purification
Recombinant Drosophila mojavensis Spastin is typically produced using yeast expression systems, which provide appropriate post-translational modifications while maintaining high protein yields . Purification generally involves affinity chromatography, with the specific tag type determined during the production process to optimize protein folding and activity .
Experimental Applications Protocol Considerations
When utilizing recombinant Drosophila mojavensis Spastin in experimental settings, several technical considerations should be addressed:
Table 3: Experimental Considerations for Recombinant D. mojavensis Spastin Use
| Application | Recommended Concentration | Buffer Conditions | Special Considerations |
|---|---|---|---|
| Microtubule Severing Assays | 50-500 nM | 20 mM HEPES pH 7.0, 1 mM MgCl₂, 1 mM ATP | Include ATP regeneration system |
| ATPase Activity Assays | 100-200 nM | 50 mM Tris-HCl pH 7.5, 20 mM KCl, 10 mM MgCl₂ | Monitor phosphate release |
| Cell Culture Studies | 1-5 μg/mL | Cell culture medium | Consider transfection efficiency |
| Structural Studies | 1-10 mg/mL | Protein-specific buffer | Remove glycerol before crystallization |
Product Specs
Note: We prioritize shipping the format currently in stock. However, should you have specific format requirements, please indicate them during order placement, and we will accommodate your request.
Note: All proteins are shipped with standard blue ice packs. If you require dry ice shipping, please contact us in advance for arrangements, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: dmo:Dmoj_GI24145
