基因,蛋白,免疫 课程之蛋白组学 5.doc

This lecture covers the use of fluorescently tagged proteins in research Fluorescent proteins allow us to visualise proteins in living cells in real time The effects of external factors such as hormones, growth factors or drugs can be studies on the labelled protein Fluorescent resonance energy transfer enables the direct interaction between two proteins to be studied within a cell Learning objectives: By the end of your revision, you should be able to explain what fluorescent tagged proteins are. You should understand the different situations in which they are useful and be able to design an experiment in which they are used. You should also appreciate the potential of fluorescence resonance energy transfer and possible pitfalls in its use. Background reading: The green fluorescent protein Tsien RY Annual Review of Biochemistry (1998) 67:509-544. Studying nuclear receptors with green fluorescence Hager GL, Methods in Enzymology (1999) 302:73-86. Fluorescence resonance energy transfer microscopy of localised protein interactions in the living cell nucleus Day RN, Periasamy A, Schaufele F, Methods (2001) 25:4-18. Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and proliferator activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription Llopis J et al., Proc. Natl. Acad. Sci. USA (2000) 97: 4363-4368. nuclear hormone receptors can be imaged in live cells and are required for transcription  Fluorescent proteins Green Fluorescent Protein (GFP) characteristics Isolated from the bioluminescent jellyfish Aequoria victoria A fluorescent protein which emits blue light with a peak near 470 nm. Structure: Size 27 kDa 11 stranded beta barrel with Central helix passing through the centre of the barrel Contains fluorophore Independent Stabile Fluorophore tripeptide Ser65-Tyr66-Gly67 Imidazolinone ring structure Absorbs: major excitation peak at 395 nm minor peak