Baculovirus Overexpression of RNA Helicase A from C. elegens and Bandshift Analysis of Its RNA Binding Properties

Shelley Batts

Date of Award

2003

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

RNA, Helicase, C.elegens, Baculovirus, RNA Helicase A, Protein Binding

Area of Concentration

Natural Sciences

Abstract

The rha-1gene from C. elegans was inserted into a pFastBac donor plasmid and transformed into E. coli colonies. These colonies were subsequently screened for correct positioning of the recombinant DNA. Positive clones were recombined into bacmid DNA which was purified and used to infect Sf9 cells which will express the RHA- 1 (RNA Helicase A) protein. Protein expression was tested with Western blot analysis, and the infected cells which proved to be overexpressing RHA- 1 were lysed, and the RHA- 1 protein was purified on a Ni- NTA resin colunm. Fractions obtained from the purification were compared by SDS-PAGE and Western blot analysis, and the fractions containing the largest concentration of protein were dialyzed. The resultant purified, dialyzed RHA- 1 protein was then tested for binding with single- and double-stranded RNA using an Electrophoretic Mobility Shift Assay (EMSA). These assays were inconclusive and suggested little to no binding was occurring. Although multiple optimization experiments testing different variable were conducted, it can be assumed that the correct binding conditions were not found.

Rights

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

This document is currently not available here.

Share

COinS