` Discovery of Green Fluorescent Protein, GFP Osamu Shimomura - - PowerPoint PPT Presentation

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` Discovery of Green Fluorescent Protein, GFP Osamu Shimomura - - PowerPoint PPT Presentation

Jun 11, 2023 358 likes •695 views

` Discovery of Green Fluorescent Protein, GFP Osamu Shimomura Ruins of the Medical College of Nagasaki, 1945 Shigeo Hayashi, 1945 Prof. Shungo Yasunaga (1911-1959) Nagasaki University Prof. Yoshimasa Hirata (1915-2000) Nagoya University

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SLIDE 1

Discovery of Green Fluorescent Protein, GFP

Osamu Shimomura

`

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SLIDE 2

Ruins of the Medical College of Nagasaki, 1945

Shigeo Hayashi, 1945

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  • Prof. Shungo Yasunaga (1911-1959)

Nagasaki University

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SLIDE 4
  • Prof. Yoshimasa Hirata (1915-2000)

Nagoya University

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SLIDE 5

Cypridina hilgendorfii

Luciferase

1 mm

Cypridina hilgendorfii

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Extraction of Cypridina luciferin

500g dried Cypridina powder Vacuu m pump

H2 gas

Red-heated Cu-fragments

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SLIDE 7

Crystals of Cypridina luciferin (1956)

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SLIDE 8

Luminescence Reaction of Cypridina Luciferin

Cypridina luciferin Oxyluciferin Light Luciferase and O2 Kishi et al., 1966

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SLIDE 9

Hikawa-Maru leaving Yokohama, Aug. 1960

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SLIDE 10

Hikawa-Maru leaving Yokohama, Aug. 1960

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SLIDE 11
  • Prof. Frank H. Johnson (1908-1990)

Princeton University

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SLIDE 12

Friday Harbor, 1961

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SLIDE 13

Aequorea aequorea

5

5 cm

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SLIDE 14
  • Univ. of Washington, Friday Harbor Laboratories, 1961
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SLIDE 15

Aequorea aequorea

In Daylight Luminescence in Dark

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Basic Strategy for the Extraction

  • f Bioluminescent Substances

A bioluminescent substance in light organs must be solubilized and extracted under a condition that reversibly inhibits the emission of light.

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Extracti

  • n of

Aequori n

Tissue of light organ (rings) Weak light pH 4 buffer Filtration Cell-free extract (pH 4) No light NaHCO3 Cell-free extract (neutral pH) Weak light Ca2+ or sea water Bright light

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SLIDE 18

Extraction of Aequorin and GFP

Rings of jellyfish (tissue of light organs) Shake in saturated (NH4)2SO4 Squeeze through gauze Filtration Granular light organs Shake in EDTA solution Filtration Crude aequorin solution Purification Aequorin and GFP

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Blue-fluorescent compound AF-350 To obtain 1 mg of AF-350, about 150 mg of aequorin is needed, requiring to collect and process 50,000 jellyfish (2.5 tons) in one summer (2,000-3,000 jellyfish per day). Urea Aequorin AF-350

2-mercaptoethanol

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Jellyfish Collectors (1974)

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  • Dr. Johnson collecting jellyfish
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. My family collecting jellyfish

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Cutter

Jellyfish Ring-cutter (made by Frank Johnson)

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Test-run of ring-cutters by Johnsons,1968

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Cutting rings

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Extraction of aequorin

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Structure Elucidation of Coelenterazine

AF-350 (Coelentramine) Coelenteramide Coelenterazine Cypridina oxyluciferin Cypridina luciferin

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Luminescence and Regeneration of Aequorin

LIGHT

465 nm

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SLIDE 29

Aequorin Luminescence with GFP

509 nm

Green Light

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GFP Crystals

Photo by Dr. Shinya Inoue

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SLIDE 31

Isolation of GFP Chromophore GFP (100 mg)

Denature at 90 °C Digest with papain Extraction with butanol at pH 1 TLC purification

Isolated chromophore (0.1 mg)

FEBS Lett. 104, 220-2 (1979)

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SLIDE 32

Chromophore of GFP

NH2 COOH G

GFP

chromophore

Ordinary Fluorescent Proteins GFP

Shimomura, 1979 Cody rt al, 1993 Amino acid residue

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SLIDE 33

Acknowledgments

I thank the many collaborators and colleagues who helped our study of aequorin and GFP, and the NSF and NIH for financial support

.