COMENIUSMeeting Green Fluorescent Protein GFP UV radiation jelly

COMENIUS-Meeting Green Fluorescent Protein - GFP UV radiation jelly fish Aequorea victoria (20 cm) green light green fluorescent protein GFP Prof. Dr. Diethard Baron University of Applied Sciences Weihenstephan-Triesdorf, D-85350 Freising Faculty for Biotechnology and Bioinformatics Teaching subjects: Biochemistry, Genetic Engineering, Immunology, English diethard. baron@web. de Prof. Dr. Diethard Baron GFP 3/9/2021 1

GFP - Nobel Prize for Chemistry in 2008 Osamu Shimomura Martin Chalfie Roger Tsien Osamu Shimomura • found GFP in jelly fish Aequorea victoria in the early 1960 s Martin Chalfie • expressed GFP in bacteria glowing green under UV radiation • labeled in the worm C. elegans proteins with GFP Roger Tsien • created by genetic engineering GFP variants emitting different fluorescent light Prof. Dr. Diethard Baron GFP 3/9/2021 2

GFP - green fluorescent protein • GFP is a protein from the jelly fish Aequorea victoria • biological function: -defence mechanism, when touched the jelly fish produces green flashing light to frighten enemies or to attract sexual partners • biotechnological application • reporter protein for gene expression • localization of proteins inside cells • to observe protein movement inside cells GFP Prof. Dr. Diethard Baron GFP 3/9/2021 3

Application of GFP protein localization cytoskeleton proteins of cancer cell localization nerve cells in the worm C. elegans with the green protein expression green bacteria colonies expressing GFP Prof. Dr. Diethard Baron GFP 3/9/2021 4

Application of GFP transgenic animals/animal development mice expressing GFP transgenic fish expressing GFP Prof. Dr. Diethard Baron GFP 3/9/2021 5

Fluorophore of GFP • the fluorescent part (fluorophore) is formed by autocatalytic reaction of the three amino acids Ser-65―Tyr-66―Gly-67 GFP • GFP contains 238 amino acids • What is the molecular mass of GFP ? ? ? Prof. Dr. Diethard Baron GFP 3/9/2021 6

Excitation and Emission • activation of GFP at 457 nm • emission at 508 nm (green light) 508 nm Prof. Dr. Diethard Baron GFP 3/9/2021 7

GFP - production of green light in jelly fish • how is GFP activated by the jelly fish in the natural environment? • in the deep-sea there is no UV lamp to activate GFP to emit green light UV radiation green light Prof. Dr. Diethard Baron GFP 3/9/2021 8

production of green light by the jelly fish 4 • • reaction partners are required Ca++ aequorin (protein, 22 k. Da) coelenterazine (432 Da) GFP in the presence of O 2 coelenterazine is bound to apoaequorin active aequorin by binding of Ca++ aequorin converts coelenterazine to activated coelenteramide returns to ground state and emits blue light (l = 469 nm) blue light activates GFP to emit green light (l = 508 nm) GFP Ca++ Prof. Dr. Diethard Baron GFP 3/9/2021 9

Fluorescent Proteins Prof. Dr. Diethard Baron GFP 3/9/2021 10

The End Prof. Dr. Diethard Baron GFP 3/9/2021 11