The Overexpression of Complexes and Subcomplexes of the Drosophila melanogaster NELF Proteins

Open Access
Author:
Hazi, Nathan Christopher
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • protein expression
  • NELF
  • protein purification
  • HPLC
  • polycistronic expression plasmid
Abstract:
The eukaryotic transcription cycle is controlled by a dynamic system of essential transcription machinery. After the preinitiation complex, including RNA Polymerase II, begins to transcribe nascent pre-mRNA, transcription elongation of many genes may be controlled by the negative elongation factor (NELF). NELF has been found to inhibit elongation in some cases, or to stall elongation, allowing a rapid induction response. NELF is a complex composed of 4 subunits: NELFA, NELFB, NELFD and NELFE. I attempted to clone all four Drosophila NELF subunits into the pST44 polycistronic expression plasmid in hopes of expressing the complex, subcomplexes and subunits in E. coli. I was able to create a tricistronic expression plasmid including dNELFD, dNELFB and HISdNELFE, but I could not subsequently incorporate the dNELFA gene into this plasmid. Each of the individual subunits and some subcomplexes were expressed at varying temperatures to determine optimal expression and solubility levels. The addition of a 6x Histidine affinity tag onto dNELFE allowed for effective affinity purification of HISdNELFE. Large-scale expression and purification of HISdNELFE was attempted, although final purification was not achieved due to truncations of the full-length protein that proved difficult to remove.