Expression and Investigation of RFP-GFP Fusion Protein

Utoft, Rasmus ; Nissen, Michael

4. term

Nanotechnology, Master

2015

2015-09-15

107

I dette speciale forsøgte vi at konstruere og udtrykke et bestemt fusionsprotein (TagRFP-T-Linker-GFP S65T), men det lykkedes ikke. I stedet flyttede vi et eksisterende TagRFP–TagGFP-gen fra plasmidet pCasper3-GR til en pET11a-vektor, hvilket skabte en konstruktion kaldet pCasper3, og udtrykte den i E. coli BL21 (DE3). Fusionsproteinet blev vurderet med SDS-PAGE (en proteingel, der viser størrelsen), absorptionsspektroskopi (hvordan lys absorberes) og fluorescensspektroskopi (hvordan det udsender lys). Gelen viste et protein af forventet størrelse. Absorptionsmaksima lå tæt på dem for de enkelte fluorescerende proteiner TagGFP og TagRFP. Fluorescensmålinger viste FRET (Förster resonans energioverførsel): når TagGFP blev exciteret, overførte det energi til TagRFP, som derefter udsendte lys—et tegn på, at de to dele er tæt nok på hinanden til at interagere. Ved at måle fluorescens under opvarmning bestemte vi smeltetemperaturer (temperaturen hvor proteinet mister sin struktur): TagGFP smeltede ved 78 °C, og TagRFP havde en smeltetemperatur på mindst 78 °C. Homologimodellering antydede en struktur med to beta-tønder forbundet af en alfa-helix-linker, med en indledende kromoforafstand omkring 5 nm (kromoforen er det lysabsorberende centrum). Yderligere analyser tydede på, at linkeren opfører sig som en tilfældig coil, og at afstanden mellem kromoforerne er større end 5 nm.

This thesis set out to build and express a specific fusion protein (TagRFP-T-Linker-GFP S65T) but was not successful. Instead, we moved an existing TagRFP–TagGFP fusion gene from the pCasper3-GR plasmid into a pET11a vector, creating a construct called pCasper3, and expressed it in E. coli BL21 (DE3). We evaluated the fusion protein with SDS-PAGE (a protein gel that shows size), absorption spectroscopy (how light is absorbed) and fluorescence spectroscopy (how it emits light). The gel showed a protein of the expected size. The absorption peaks were close to those of the individual fluorescent proteins TagGFP and TagRFP. Fluorescence measurements revealed FRET (Förster resonance energy transfer): when TagGFP was excited it transferred energy to TagRFP, which then emitted light—evidence that the two parts are close enough to interact. By scanning fluorescence as the temperature increased, we estimated melting temperatures (the point where the protein loses its structure): TagGFP melted at 78 °C and TagRFP at least at 78 °C. Homology modeling suggested a structure of two beta-barrel domains connected by an alpha-helix linker, with an initial chromophore distance of about 5 nm (the chromophore is the light-absorbing core). Additional analyses indicated that the linker likely behaves as a random coil and that the chromophores are more than 5 nm apart.

[This abstract was generated with the help of AI]

Fluorescent protein ; GFP ; RFP ; FRET ; Master

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