275018 Cloning Nuclear Hormone Receptors to Develop Biosensors Using the Gibson Assembly

Wednesday, October 31, 2012: 8:48 AM
Westmoreland West (Westin )
Miriam Shakalli Tang1, Richard A. Lease2 and David Wood1, (1)Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, (2)Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH

Cloning Nuclear Hormone Receptors to develop Biosensors using the Gibson Assembly

Miriam Shakalli Tang, Richard A. Lease, David W. Wood

Nuclear hormone receptor (NHR) protein domains comprise one of the largest classes of protein targets for drug ligands because they can bind tightly to a wide variety of endocrine disruptors in addition to their native hormones.  We are developing high-throughput methods for detection and characterization of hormone-like compounds, to be used in applications such as toxicology screening, insecticide development, and drug discovery. Current compound screening strategies for hormone mimics are complex, time consuming, and expensive. To address this, we have developed a bacterial biosensor containing a genetically engineered reporter protein.  This reporter protein incorporates a hormone ligand-binding domain (LBD) of an animal nuclear hormone receptor within a bacterial intein-protein platform technology. These biosensors can detect compounds that target NHRs, where the readout is a simple growth assay.  These biosensors can distinguish positive or negative effects of a test compound on the receptor's activity, as well as agonist versus antagonist activities.

The goal of this project is to optimize the rapid construction of nuclear hormone binding sensors using the Gibson Assembly method, and to apply the Gibson method to the development of protein biosensors based on NHRs from Aedes aegypti (mosquito vector for dengue fever) and Tribolium confusum (red flour beetle).  Gibson Assembly is a robust in vitro recombinant technology, which allows efficient construction of recombinant fusion proteins.  In this case, the Gibson strategy allowed the insertion of the LBD of the insect NHR gene into a larger fusion protein gene.  The fusion protein gene includes solubilization and stabilization domains (maltose-binding protein plus an intein linker domain), as well as a thymidylate synthase (TS) enzyme domain to generate the bacterial growth phenotype. The resulting reporter protein links binding activity of the LBD to TS activity and subsequence bacterial growth phenotype.  The constructed sensors were subsequently tested to discern the presence of hormones or hormone-like compounds through changes in growth phenotype of TS knockout E. coli strains in thymineless media. Following the success of the Gibson Assembly in cloning NHR biosensors, its capability of joining DNA fragments will be harnessed for the rapid (HTS) production of recombinant protein products and for screening of endocrine disruptor ligands as potential insecticides.

Extended Abstract: File Not Uploaded