Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Beatrice Edjah - Georgia State University
Zinc fingers (ZnF) are small protein modules in which various combinations of cysteines and histidines coordinate a zinc ion. ZnF proteins are highly abundant and serve as transcription factors in multiple biological processes. This study seeks to characterize an isotopically (13C, 15N) labeled ZnF29G29R, a ZnF protein that has been found to bind to the RREIIB loop of the human immunodeficiency virus (HIV) with a high affinity as shown from a previously conducted study by Mishra et al 1). Binding of the viral regulatory protein REV to the RREIIB loop initiates the export of viral RNA from the nucleus and into the cytoplasm where new HIV virions are packaged. Interference of the REV binding caused by the ZnF29G29R inhibits the export of viral RNA in the HIV life cycle and is therefore of therapeutic interest. The labeled ZnF29G29R was initially expressed as the fusion protein ZnF+Thioredoxin+His6-tag in an optimized minimal media E.coli culture. Using a nickel affinity column, the fusion proteins were extracted from the lysate and dialyzed. The ZnFs were cleaved from the Thioredoxin+His6-tag complexes using the enzyme enterokinase and were then extracted using a cation ion exchange column. Size exclusion chromatography allowed for further purification of the proteins. Using a UV-Visible spectrophotometer, it was determined that a 250mL minimal media expression produced 6-9 mg of protein. The purity of the proteins were assessed using SDS-PAGE gels and the proper folding of the proteins was determined by observing the Phe14 and His27 δ2H chemical shifts using1H NMR. Future studies will entail studying the complex formed between the labeled ZnF and labeled RREIIB RNA using multidimensional NMR.
References: 1) Mishra, S. H.; Shelley, C. M.; Barrow, D. J., Jr.; Darby, M. K.; Germann, M. W. 2006. Solution structures and characterization of human immunodeficiency virus Rev responsive element IIB RNA targeting zinc finger proteins. Biopolymers. 83(4): 352-64.
Funder Acknowledgement(s): This study was supported by a grant from NIH and the Georgia Cancer Coalition.
Faculty Advisor: Markus Germann, email@example.com
Role: I conducted all of the research methods.