Expression of recombinant mouse spastin in E. coli

Yujuan Wang

doi:10.1051/e3sconf/202018504058

Abstract

Mutations of the gene SPAST that encodes a microtubule severing enzyme, spastin, are the most frequent cause of Hereditary spastic paraplegia (HSP) disease. HSP is heterogeneous group of inherited neurodegenerative disorders characterized predominantly by progressive lower limb spasticity and weakness. Spastin belongs ATPase associated with various cellular activities (AAA) protein family and catalyzes microtubule severing. Spastin in mouse and human are highly identical in protein sequence and several spastin mutation models in mice have been generated in order to evaluate the significance of spastin loss-of-function in mammals. Expression and purification of spastin and the mutant variants determined in patients will facilitate the structure-function relationship study of spastin. Here I systemically optimized the expression condition of a truncated version of mouse spastin in E. coli. The recombinant protein and a mutant were further purified for ATPase activity assay.

Highlights

In human, mutations in the SPAST gene that encodes spastin are the most common cause of Hereditary Spastic Paraplegia (HSP)
HSP is a group of neurodegenerative diseases with significant clinical and genetic heterogeneity, characterized by predominantly, but not exclusively, slow progressive weakness of the lower limbs and spastic paraplegia[1]
Spastin is a member of ATPase associated with various cellular activities (AAA) protein family

Summary

Introduction

Mutations in the SPAST (previously known as SPG4) gene that encodes spastin are the most common cause of Hereditary Spastic Paraplegia (HSP). Human SPAST gene locus on 2p22.3 contains 17 exons and encodes a microtubule severing enzyme, spastin of 616 amino acids. AAA domain between residues 342 to 599 is located on the C-terminus of spastin protein and is involved catalyzing microtubule cleavage[2]. Spastin contains microtubule-binding domain (MTBD), hydrophobic domain (HD) that forms the hairpin structure, and the domain of microtubule interacting and trafficking (MIT) domain. MTBD, spanning residues 270 to 328, promotes the binding of spastin and microtubules before severing microtubules[2]. Spastin performs the microtubule severing activity depending on the disordered and negatively charged C-terminal tails of the tubulin. Study has revealed that spastin use its pore loops to tug the C-terminal tail of tubulin and generates a mechanical force accompanied with hydrolysis of ATP[1,4]

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Results

Conclusion