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Overexpression and Characterization of the rquA Gene Product Involved in the Biosynthesis of Rhodoquinone
Author(s) -
Zander Alison,
Shepherd Jennifer
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.919.1
Subject(s) - biosynthesis , gene , rhodospirillum rubrum , biology , genome , gene product , biochemistry , computational biology , enzyme , gene expression
Infections caused by parasitic helminths are sometimes referred to as “the great neglected tropical diseases.” Endemic to many countries, including the United States, parasitic helminths are at the origin of the diseases most commonly diagnosed in developing countries. Affecting both humans and livestock, these helminths are the target of extensive research as their genome is further elucidated. The drug products that currently exist to combat infections are subject to parasitic resistance; therefore, the race to discover different drug‐targets specific to these parasites is essential. In an effort to develop an answer to this problem, we have been researching the rhodoquinone (RQ) biosynthetic pathway. RQ is the final electron transporter in the anaerobic fumarate reductase pathway, which occurs in the mitochondrial and plasma membranes. Although its biosynthetic pathway is still largely unknown, it has been discovered that the gene known as rquA is required for RQ biosynthesis in the model organism, Rhodospirillum rubrum . Expression and purification of the gene product of rquA (RquA) could provide a target for inhibition of RQ biosynthesis. Additionally, determining the crystalline structure and any proteins that are in a complex with RquA could further information in development of a novel drug. After isolation and purification of RquA from overexpression in E. coli , a pull‐down experiment was performed using nickel‐coated magnetic beads to bind the protein (containing a hexahistidine tag). Following this, the bead protein complex was combined with lysate collected from solubilized wild‐type R. rubrum membranes. Providing these conditions for the protein allowed RquA to complex with any other proteins that it may interact with in its normal state. Upon SDS‐PAGE gel electrophoresis, and staining with Coomassie blue, there was evidence of at least 4 proteins that appear to be associated with RquA. Support or Funding Information National Institutes of Health (1R15GM096398‐01) and Howard Hughes Medical Institute (award to Gonzaga University)