Expression and Purification of Corticosterone Binding Globulin in E. colifor Quantification

• Published in: The FASEB journal Vol. 36
• Main Authors: Ridley, Lauryn A., Malisch, Jessica L., Odunuga, Odutayo O., Mertz, Pamela S.
• Format: Journal Article
• Language: English
• Published: United States The Federation of American Societies for Experimental Biology 01-05-2022
• DOI: 10.1096/fasebj.2022.36.S1.R2426
• EISSN: 1530-6860
• ISSN: 0892-6638

Corticosterone (CORT) is analogous to the mammalian stress hormone cortisol and is involved in the acute stress response in birds. Birds can be used as a model of the stress response due to the evolutionarily conserved activation of the hypothalamic‐pituitary‐adrenal (HPA) axis and sympathetic adrenomedullary (SA) system across vertebrate species. Corticosterone binding globulin (CBG) is a protein that binds and carries approximately 80% of corticosterone in the plasma in both avian and mammalian species. There are limitations in the most common technique for quantification of CBG due to the use of radioactive binding assay. The ultimate goal of this project is to develop a robust and safe method to quantify CBG levels. This reports the first part of that goal – the expression and purification of variously‐tagged zebra finch CBG from E. coli. CBG was expressed either as histidine‐tagged or in fusion with glutathione‐S‐transferase (GST) or maltose‐binding protein (MBP). Purified CBG was confirmed and assessed by SDS‐PAGE and western blot using antiserum to zebra finch CBG. Following purification, circular dichroism will be used to analyze the secondary structure of the recombinant CBG protein. The project aims to develop an ELISA‐based method to measure the amount of CBG in samples.