Maximizing expression of flag-tagged estrogen receptor beta and development of a dual expression baculoviral system for the expression of estrogen receptor alpha and beta.

摘要

It is well-established that estrogen is critical for breast cancer development. The effects of estrogen in the cell are mediated through a member of the steroid/nuclear receptor superfamily, the estrogen receptor (ER). There are two subtypes of nuclear ER: ERalpha and ERbeta. ERs form ERalpha/ERalpha or ERbeta/ERbeta homodimers or, in cells that express both subtypes, ERalpha/ERbeta heterodimers. The overall goal of the project described in this thesis was to generate baculoviral expression vector systems for the expression of Flag-tagged hERbeta1 homodimers and Myc-hERalpha/Flag-hERbeta1 heterodimers. These proteins were generated using a baculoviral expression system that inserts a target gene into a baculovirus. Subsequent infection of Sf21 insect cells with the baculovirus produces target protein. In this thesis I will demonstrate that I have generated Flag-hERbeta1 and coexpression of Myc-hERalpha and Flag-hERbeta1 using separate viruses. Also, the K d of Flag-hERbeta1 for different estrogen response elements was determined using Electrophoretic Mobility Shift Assays.