Expression, Purification and Metal Utilization of Recombinant Soda From Borrelia Burgdorferi
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.
Activity was only possible when cells accumulated extremely high levels of manganese that exceeded cellular iron. Moreover, there was no evidence for iron inactivation of the SOD. B. burgdorferi SodA shows strong overall homology with other members of the Mn-SOD family, but computer-assisted modeling revealed some unusual features of the hydrogen bonding network near the enzyme's active site. The unique properties of B. burgdorferi SodA may represent adaptation to expression in the manganese-rich and iron-poor environment of the spirochete.
The causative agent of Lyme disease is the spirochetal pathogen Borrelia burgdorferi, which is transmitted to humans through the bite of an infected Ixodes sp. tick (1). There is currently very little information available on the tissue-specific host-pathogen interactions that lead to pathological manifestations of B. burgdorferi infection. This pathogen's ability to colonize mammals is dependent on its capacity to rapidly alter gene expression in response to highly disparate environmental signals following transmission from infected ticks (Tamborero S, Vilar M, 2011).
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.
Tamborero S, Vilar M, Martinez-Gil L, Johnson AE, Mingarro I. 2011. Membrane insertion and topology of the translocating chain-associating membrane protein (TRAM). J. Mol. Biol.
Saaf A, Wallin E, von Heijne G. 1998. Stop-transfer function of pseudo-random amino acid segments during translocation across prokaryotic and eukaryotic membranes. Eur. J. Biochem. 251:821–829
Martinez-Gil L, Bano-Polo M, Redondo N, Sanchez-Martinez S, Nieva JL, Carrasco L, Mingarro I. 2011. Membrane integration of poliovirus 2B viroporin. J. Virol.
Raibaud S, Schwarz-Linek U, Kim JH, Jenkins HT, Baines ER, Gurusiddappa Hook S M, Potts JR. 2005. Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes. J. Biol. Chem.
Emsley J, Cruz M, Handin R, Liddington R. 1998. Crystal structure of the von Willebrand factor A1 domain and implications for the binding of platelet glycoprotein Ib. J. Biol. Chem.