Essay sample

Expression, Purification and Metal Utilization of Recombinant Soda From Borrelia Burgdorferi

Free ideas for

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.

Free ideas for

The Lyme disease pathogen Borrelia burgdorferi represents a novel organism in which to study metalloprotein biology in that this spirochete has uniquely evolved with no requirement for iron. Not only is iron low, but we show here that B. burgdorferi has the capacity to accumulate remarkably high levels of manganese. This high manganese is necessary to activate the SodA superoxide dismutase (SOD) essential for virulence. Using a metalloproteomic approach, we demonstrate that a bulk of B. burgdorferi SodA directly associates with manganese, and a smaller pool of inactive enzyme accumulates as apoprotein. Other metalloproteins may have similarly adapted to using manganese as co-factor, including the BB0366 aminopeptidase. Whereas B. burgdorferi SodA has evolved in a manganese-rich, iron-poor environment, the opposite is true for Mn-SODs of organisms such as Escherichia coli and bakers' yeast. These Mn-SODs still capture manganese in an iron-rich cell, and we tested whether the same is true for Borrelia SodA. When expressed in the iron-rich mitochondria of Saccharomyces cerevisiae, B. burgdorferi SodA was inactive

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.

Free ideas for

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays (Saaf A, Wallin E, 1998). Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. Lyme disease is a multisystemic disorder that leads to arthritis in 60% of cases, carditis in 10% of untreated adults, and other neurological symptoms

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).

Free ideas for

Summing up, 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.

Free ideas for

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.

Was this essay example useful for you?

Do you need extra help?

Order unique essay written for you
essay statistic graph
Topic Popularity