Purified Aryl Hydrocarbon Receptor Nuclear Translocator in Increasing Expression of the Aryl Hydrocarbon Receptor Complex
Poster Number
32
Introduction/Abstract
The deletion construct of aryl hydrocarbon receptor (AhR), CΔ553 dimerizes with the deletion construct of aryl hydrocarbon receptor nuclear translocator (Arnt), CΔ418, forming the AhR/Arnt complex. It has been observed in the preceding studies that denatured CΔ418 was successful, as determined by gel shift assay, in assisting the refolding of CΔ553 but only in buffers with high molar concentration of arginine, thereby limiting the ability of the AhR/Arnt complex.
Purpose
To determine if using native purification of CΔ418 will assist in refolding denatured CΔ553, allowing interaction of the AhR/Arnt complex.
Method
CΔ418 was enriched using native affinity purification whereas CΔ553 was purified under denatured affinity purification using 6M guanidine hydrochloride. CΔ553 was combined with CΔ418, followed by protein refolding via limited dialysis to gradually get rid of denaturant and introduce arginine into the solution. Silver staining was used to determine the yield of the refolded CΔ418 and CΔ553. Gel Shift Assay was performed to determine the DNA-binding ability of the AhR/Arnt complex.
Results
Concentrated CΔ418 is able to refold CΔ553 in buffers containing no denaturant and arginine. Conclusion: The interaction between CΔ418 and CΔ553 observed in assay buffer containing no arginine or denaturant shows that native purified CΔ418 appears to better assist in refolding CΔ553.
Significance
The successful refolding of CΔ553 by purified CΔ418 will allow structural study of AhR. For example, future cross-linking reactions in order to further understand the interactional, structural, and functional relationship between CΔ418 and CΔ553.
Location
DeRosa University Center, Stockton campus, University of the Pacific
Format
Poster Presentation
Purified Aryl Hydrocarbon Receptor Nuclear Translocator in Increasing Expression of the Aryl Hydrocarbon Receptor Complex
DeRosa University Center, Stockton campus, University of the Pacific
The deletion construct of aryl hydrocarbon receptor (AhR), CΔ553 dimerizes with the deletion construct of aryl hydrocarbon receptor nuclear translocator (Arnt), CΔ418, forming the AhR/Arnt complex. It has been observed in the preceding studies that denatured CΔ418 was successful, as determined by gel shift assay, in assisting the refolding of CΔ553 but only in buffers with high molar concentration of arginine, thereby limiting the ability of the AhR/Arnt complex.
