Genetically Engineering Plasmid Expression Vectors to Study Cellular Localization of Reporter Proteins
Date of Award
Bachelor of Science
Major or Concentration
Proteins can enter the nucleus by simple diffusion or by a facilitated transport mechanism known as nuclear import. Facilitated transport requires the presence of a nuclear localization signal (NLS), a relatively short, eight amino acid peptide embedded in the primary structure of the protein (Alberts et al. 2008). The long-term goal of this research project is to create recombinant plasmid expression vectors that can be used by future undergraduate students to study the cellular localization of proteins, including an expression vector to study nuclear import and the nuclear localization signal (NLS). Due to its relatively high molecular weight and its natural fluorescence, a fusion protein consisting of multiple copies of green flourescent protein (GFP) linked to an NLS is an ideal reporter protein for nuclear localization studies. The specific objective of this research study is to create plasmids of various molecular weights containing zero NLSs. The three NLSs were removed from the two plasmids created by Krumpos et al., creating plasmids containing two copies of GFP open reading frames (ORFs) and the GFP ORFs but lacking the three NLSs. The expression of these plasmids in mammalian cells will result in the synthesis of a fusion protein cotaining 2 copies of the GFP protein (total molecular weight approximately 54,000 Daltons) and a fusion protein containing 3 copies of the GFP protein (total molecular weight approximately 81,000 Daltons), respectively. The Lack of NLSs in these expressed proteins will prevent them utilizing the classical nuclear import mechanism and limit them to entry into the nucleus via simple diffusion.
Thomas, Rachel Ann, "Genetically Engineering Plasmid Expression Vectors to Study Cellular Localization of Reporter Proteins" (2016). Student Research Submissions. 75.