(447b) Cloning Nuclear Hormone Receptors to Develop Biosensors Using the Gibson Assembly

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.