22 May 2012

Super Luciferase

Russian scientists have obtained a mutant protein that emits a lot of light

Alexey Shabelsky, Informnauka

To understand what is happening inside the cell in real time, scientists often use bioluminescent reporter proteins with the beautiful name "luciferase" (from the word "lucifer" – a light carrier). A team of Russian researchers from the Institute of Biophysics SB RAS followed the same path, trying to spy on intracellular processes.

Siberian scientists have cloned a gene encoding luciferase of marine copepods (crustaceans) and modified this protein responsible for the glow of these organisms using genetically engineered methods.

By studying the luminescence intensity of such molecules, the researchers demonstrated for the first time that modified proteins emit five times more light than their natural, unchanged counterparts. The results of the work are published in Biochemical and Biophysical Research Communications (Markova et al., High-active truncated luciferase of copepod Metridia longa).

Perhaps today there is not a single large pharmaceutical company that would not use bioluminescent monitoring systems in preclinical trials of medicines. In order to take these experiments to a new level, it is necessary to understand even more clearly what processes are taking place inside the cell in real time. This is quite difficult, so there are not many scientific groups in the world that set themselves such tasks. There is one team each in the USA, Japan and Russia. Scientists of the Institute of Biophysics SB RAS are engaged in these studies here. They decided to look into the cell using modified luminous luciferase proteins. Their choice was based on the fact that the laboratory had previously worked with the metridium gene encoding luciferase of these organisms.

"In some cases, long-term monitoring of intracellular events using luciferases in real time is fraught with certain difficulties, especially if firefly luciferase is used as a reporter protein, whose bioluminescent reaction requires oxygen, ATP and magnesium ions in addition to a specific luciferin substrate," explains Evgeny Vysotsky, head of the Institute's photobiology laboratory, head of the study. – It is not easy to maintain such a large number of reaction components at a constant level inside the cell. Metridia longa luciferase lacks these disadvantages, since bioluminescence requires only the luminous protein itself, the substrate – coelenterazine, and oxygen. In addition, this luciferase belongs to the class of secreted enzymes, i.e. after synthesis in the cell, the protein is "released" by the cell into the surrounding space. Thus, it allows monitoring of intracellular events with high sensitivity without destroying the cells and tissues themselves."

In the course of research led by Evgeny Vysotsky, scientists cloned a gene encoding a protein from marine copepods Metridia longa, which catalyzes the reaction with light emission. Then, with the help of genetic manipulations, they purposefully changed the normal position of amino acids in the protein, resulting in mutant forms of the protein. The nucleotide sequences encoding such proteins were inserted into plasmids – circular DNA molecules twisted into a superspiral and capable of self-replication in the host cell. Plasmids, in turn, were introduced into the cells of Escherichia coli bacteria, after which these cells became capable of synthesizing metridium luciferase. Scientists isolated mutant forms of protein from them, purified them and thoroughly examined them.

As a rule, protein molecules, or in another way enzymes, consist of various functional sites, sometimes not directly involved in the process of catalysis. In the case of luciferase from Metridia longa, amino acids located in the so-called N-terminal part of luciferase did not affect the bioluminescent activity. In fact, a simple truncation of the N-terminal parts (the side of the molecule ending with the amino group) of metridium luciferase led to a significant, five-fold increase in the intensity of the glow and the effectiveness of the bioluminescent reaction. In addition, the authors have shown that metridium luciferase with an amino acid removed in a certain position is well suited for the role of a reporter protein in mammalian cells. Luminescent analysis performed using such proteins shows greater sensitivity than a similar analysis using natural enzymes.

As Evgeny Vysotsky notes, for secreted luciferases, an increase in activity due to enzyme truncation has been demonstrated for the first time.

The authors of the work participate in the project of megagrants of the second wave to attract leading scientists to work in Russian universities. (Grants 16.512.11.2141 and 64987.2010.4 of the Ministry of Education and Science of the Russian Federation). As part of this grant, Osamu Shimomura, a Japanese Nobel Prize winner in Chemistry for discoveries in the field of fluorescent proteins, will create a laboratory at Siberian Federal University.

Portal "Eternal youth" http://vechnayamolodost.ru22.05.2012

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