Expression, purification and metal utilization of recombinant SodA from Borrelia burgdorferi

G Brown, A H Broxham, S E Cherrington, D. C. Thomas, A Dyer, L Stejskal, R J Bingham

Research output: Contribution to journalArticle

Abstract

Borrelia are microaerophilic spirochetes capable of causing multisystemic diseases such as Lyme disease and Relapsing Fever. The ubiquitous Fe/Mn-dependent superoxide dismutase (SOD) provides essential protection from oxidative damage by the superoxide anion. Borrelia possess a single SOD enzyme - SodA that is essential for virulence, providing protection against host-derived reactive oxygen species (ROS). Here we present a method for recombinant expression and purification of Borrelia burgdorferi SodA in E. coli. Metal exchange or insertion into the Fe/Mn-SOD is inhibited in the folded state. We therefore present a method whereby the recombinant Borrelia SodA binds to Mn under denaturing conditions and is subsequently refolded by a reduction in denaturant. SodA purified by metal affinity chromatography and size exclusion chromatography reveals a single band on SDS-PAGE. Protein folding is confirmed by circular dichroism. A coupled enzyme assay demonstrates SOD activity in the presence of Mn, but not Fe. The apparent molecular weight determined by size exclusion corresponds to a dimer of SodA; a homology model of dimeric SodA is presented revealing a surface Cys distal to the dimer interface. The method presented of acquiring a target metal under denaturing conditions may be applicable to the refolding of other metal-binding proteins.

LanguageEnglish
Article number105447
Number of pages6
JournalProtein Expression and Purification
Volume163
Early online date1 Jul 2019
DOIs
Publication statusE-pub ahead of print - 1 Jul 2019

Fingerprint

Borrelia burgdorferi
Superoxide Dismutase
Borrelia
Metals
Relapsing Fever
Spirochaetales
Lyme Disease
Protein Folding
Enzyme Assays
Circular Dichroism
Affinity Chromatography
Superoxides
Gel Chromatography
Virulence
Polyacrylamide Gel Electrophoresis
Reactive Oxygen Species
Carrier Proteins
Molecular Weight
Escherichia coli
Enzymes

Cite this

Brown, G ; Broxham, A H ; Cherrington, S E ; Thomas, D. C. ; Dyer, A ; Stejskal, L ; Bingham, R J. / Expression, purification and metal utilization of recombinant SodA from Borrelia burgdorferi. In: Protein Expression and Purification. 2019 ; Vol. 163.
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Expression, purification and metal utilization of recombinant SodA from Borrelia burgdorferi. / Brown, G; Broxham, A H; Cherrington, S E; Thomas, D. C.; Dyer, A; Stejskal, L; Bingham, R J.

In: Protein Expression and Purification, Vol. 163, 105447, 01.11.2019.

Research output: Contribution to journalArticle

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T1 - Expression, purification and metal utilization of recombinant SodA from Borrelia burgdorferi

AU - Brown, G

AU - Broxham, A H

AU - Cherrington, S E

AU - Thomas, D. C.

AU - Dyer, A

AU - Stejskal, L

AU - Bingham, R J

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AB - Borrelia are microaerophilic spirochetes capable of causing multisystemic diseases such as Lyme disease and Relapsing Fever. The ubiquitous Fe/Mn-dependent superoxide dismutase (SOD) provides essential protection from oxidative damage by the superoxide anion. Borrelia possess a single SOD enzyme - SodA that is essential for virulence, providing protection against host-derived reactive oxygen species (ROS). Here we present a method for recombinant expression and purification of Borrelia burgdorferi SodA in E. coli. Metal exchange or insertion into the Fe/Mn-SOD is inhibited in the folded state. We therefore present a method whereby the recombinant Borrelia SodA binds to Mn under denaturing conditions and is subsequently refolded by a reduction in denaturant. SodA purified by metal affinity chromatography and size exclusion chromatography reveals a single band on SDS-PAGE. Protein folding is confirmed by circular dichroism. A coupled enzyme assay demonstrates SOD activity in the presence of Mn, but not Fe. The apparent molecular weight determined by size exclusion corresponds to a dimer of SodA; a homology model of dimeric SodA is presented revealing a surface Cys distal to the dimer interface. The method presented of acquiring a target metal under denaturing conditions may be applicable to the refolding of other metal-binding proteins.

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