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Protein Yields Vivid Images

Photonics.com
Aug 2007
MOSCOW, Aug. 30, 2007 -- A new red fluorescent protein derived from a brilliant red sea anemone purchased in a Moscow pet shop reveals body tissues more vividly than other fluorescent proteins in use today. The protein's developers said the glow it emits can render cancers and other target tissues easily visible in living animals.

The development of the red protein, which the researchers call Katushka, was reported by Howard Hughes Medical Institute (HHMI) international research scholars Andrey Zaraisky, Sergey A. Lukyanov, and their colleagues Aug. 26 in the online version of Nature Methods. Principal development of Katushka was carried out by Dmitry Chudakov and colleagues in Lukyanov's laboratory at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry in Moscow; Zaraisky and his colleagues are also at the institute.Katushka.jpg
Transgenic 2.5-month-old African clawed frogs (Xenopus laevis) expressing Katushka (left) and DsRed-Express (right) under the control of muscle actin promoter. The frogs are shown from the dorsal side under white light. (Image courtesy Andrey Zaraisky)
According to the researchers, Katushka solves a major problem in the field of fluorescent reporter proteins. Fluorescent proteins have become invaluable research tools for labeling specific genes and tissues. This labeling permits researchers to follow gene activity visually or to track cellular development. However, there is no tracer that glows brightly in a particular “window” of the far-red spectrum favorable for maximally penetrating living tissues. Thus, such proteins were not practical for optically imaging tagged genes, cells or tissues in whole animals.

The development of Katushka might never have happened had it not been for Lukyanov's shrewd bargaining skills. Visiting a Moscow pet shop, he saw a brilliant red sea anemone in its aquarium. Sensing that the anemone's vivid red coloring might provide the blueprint for a new biological tracer, he tried to buy it. Told that it was already sold, he nevertheless persisted, outbidding the potential buyer and procuring the creature.

Back in Lukyanov's laboratory, Chudakov and his coworkers isolated the red protein from the anemone and then developed an enhanced version, which they named turbo red fluorescent protein (TurboRFP). The protein comprised a string of identical protein subunits, so the researchers also developed a single-unit monomeric version, which they called TagRFP.

Although TurboRFP was twice as bright as a comparable red protein, DsRed2, the researcher set out to improve its brightness in the far-red window. They created about 100,000 variants of the gene for TurboRFP, screening the resulting proteins for high brightness in the near-infrared. This screening yielded a highly bright variant protein, which they further optimized by randomly mutating the gene and selecting the brightest protein.

They named this protein Katushka -- a Russian diminutive for the name Ekaterina -- after coauthor Ekaterina Merzlyak, in recognition of her work developing TurboRFP and TagRFP. Katushka proved to be up to tenfold brighter in the far red, compared to the spectrally close fluorescent proteins HcRed and mPlum. mPlum was developed in 2004 by HHMI investigator Roger Y. Tsien at the University of California, San Diego, a leader in the study of fluorescent proteins for cell biology research. The researchers also generated a monomeric version of Katushka, called mKate, which is useful for molecular labeling of proteins.

Zaraisky and his colleagues tested Katushka's properties as a tracer by introducing it into living cells in culture, as well as into the muscle cells of the African clawed frog Xenopus laevis, widely used in research. They found the protein to be highly visible and nontoxic.

“The key feature of Katushka that makes it valuable as a fluorescent marker is the unique combination of high brightness, far-red emission and fast rate of the chromophore maturation,” said Zaraisky. “As far as I know, none of the known fluorescent proteins demonstrates this combination of properties.”

Early appearance of the Katushka protein in tissues is particularly important, said Zaraisky. “It's desirable to detect transgenic expression at the earliest stage of embryonic development to be able to investigate early events of embryogenesis,” he said. “This means that you need to use the fastest-maturing and brightest fluorescent protein as a reporter. At the same time, since frog embryos are not transparent at early stages, your fluorescent reporter should emit as far in the infrared as possible, because the longer wavelength can more easily pass through living tissues.”

As an added benefit, the researchers found that the Katushka protein persisted in labeled tissues as the frogs reached adulthood. “We hoped that Katushka could be visible in adult frogs, but it was a surprise that it has appeared so bright,” Zaraisky said.

Zaraisky sees broad uses for Katushka in research. “We believe that Katushka has a big potential in cancer research, because it could significantly simplify some experiments that should be done only at the whole-body level. It would be possible, for example, to generate model lines of mice that would specifically express the Katushka transgene in cells of different types of cancer. Then, these lines of mice could be used to test influence of different drugs on progression of cancer metastases.

“Also, Katushka is an excellent choice as a living reporter for experiments in developmental biology, especially for such semitransparent models as frog and early mouse embryos,” he said. For example, Zaraisky and his colleagues plan to use the frog embryos generated in their analysis of Katushka's properties to study muscle cell differentiation.

According to Chudakov, further improvements in Katushka are likely. “The palette of basic fluorescent proteins is currently almost complete, the only gap remaining in the very far-red part of the visible spectrum,” he said. “We hope that further development of Katushka and mKate will finally fill the gap.”

For more information, visit: www.hhmi.org


GLOSSARY
cell
1. A single unit in a device for changing radiant energy to electrical energy or for controlling current flow in a circuit. 2. A single unit in a device whose resistance varies with radiant energy. 3. A single unit of a battery, primary or secondary, for converting chemical energy into electrical energy. 4. A simple unit of storage in a computer. 5. A limited region of space. 6. Part of a lens barrel holding one or more lenses.
photonics
The technology of generating and harnessing light and other forms of radiant energy whose quantum unit is the photon. The science includes light emission, transmission, deflection, amplification and detection by optical components and instruments, lasers and other light sources, fiber optics, electro-optical instrumentation, related hardware and electronics, and sophisticated systems. The range of applications of photonics extends from energy generation to detection to communications and...
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