Expression and Investigation of RFP-GFP Fusion Protein

Student thesis: Master Thesis and HD Thesis

  • Rasmus Utoft
  • Michael Nissen
4. term, Nanotechnology, Master (Master Programme)
In this master’s thesis attempts were made at constructing and expressing a TagRFP-T-Linker-GFP S65T
fusion protein. These attempts were unsuccessful. Instead, the TagRFP-TagGFP fusion protein gene was
transferred from pCasper3-GR to a pET11a vector using PCR, constructing pCasper3. The pCasper3
was expressed in E. coli BL21 (DE3). The fusion protein was analyzed using SDS-PAGE, fluorescence
spectroscopy and absorption spectroscopy. SDS-PAGE showed expression of a correct size protein. Absorption spectroscopy showed maxima close to those of TagGFP and TagRFP. Fluorescence spectroscopy
showed that FRET effects were responsible for the TagRFP fluorescence during TagGFP excitation. Temperature scan fluorescence spectroscopy was used to find the melting temperature of TagGFP which was
78 ◦C. The melting temperature of TagRFP was found to be at least 78 ◦C. Homology modelling found
that the structure of the fusion protein was two β-barrels connected by an α-helix linker. The distance between the chromophore was found to be 5 nm. Further studies suggested that the linker formed a random
coil structure and that the distance between the chromophores was more than 5 nm.
Publication date15 Sept 2015
Number of pages107
ID: 218879063