Scientists get their kicks from extreme gel electrophoresis

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  • Published: Jul 22, 2006
  • Author: Jon Evans
  • Channels: Electrophoresis
thumbnail image: Scientists get their kicks from extreme gel electrophoresis

Micro-organisms that are able to thrive in extreme environments, known as extremophiles, are interesting both from a scientific and an industrial perspective. Understanding how extremophiles are able to survive such inhospitable conditions should help to reveal the limits of life on Earth, including how it got going in the first place, and also uncover unique biological processes for use in industrial biotechnology. A team of US researchers has recently furthered this understanding by conducting one of the first proteomic studies of the extremophile Sulfolobus solfataricus.

This species of extremophile is able to withstand extremes of both temperature (up to nearly 100°C) and pH (down to 0.5), and grows best at 80°C and pH 3. It is fairly common and has been found growing in geothermal springs all over the world, including in the US, Iceland, New Zealand and El Salvador.

Along with a number of other species of extremophile, it recently had its genome sequenced. This revealed genes coding for almost 3,000 different proteins, of which around 45% had an unknown function. To try to find out more about these 3,000 proteins, the team of US researchers led by Edward Dratz from Montana State University, Bozeman, turned to 2D gel electrophoresis (2-DE).

They conducted a number of different separations using different variations of 2-DE in order to uncover as many proteins as possible. For instance, they divided the proteins extracted from S. solfataricus cells into those that were soluble or insoluble in a Tris-HCl buffer. This basically meant that the researchers could analyse those proteins from the interior of the cell separately to those from the cell membrane, which tend to be much more hydrophobic - and thus less soluble - than proteins from the cell interior.

They also used two different buffers to try to keep the proteins soluble during the gel-based separation. These buffers differed mainly in terms of how reducing they were and the researchers hoped that using both of them would maximise the number of proteins that could be separated on the gel.

In total, Dratz and his colleagues resolved around 1300 individual spots on all the gels. Using peptide mass fingerprinting, the researchers identified 867 of these spots and found that they represented 324 different proteins. Of these, they found 274 proteins in the Tris-soluble fraction and 100 proteins in the Tris-insoluble fraction, with 50 proteins present in both fractions.

In accordance with their prior assumption, the researchers found that the proteins in the Tris-soluble fraction were dominated by those normally present in the cell interior. These included proteins involved in DNA replication, translation and transcription, as well as transport and energy production. In contrast, the Tris-insoluble fraction was dominated by membrane-associated proteins such as ATPases, ATP synthetases and ABC transporters.

But the researchers were obviously especially interested in proteins that are unique to extremophiles, and they found quite a few. These included several stress-responsive proteins, such as heat-shock proteins and oxidative stress-related proteins. Even more interestingly, they identified an enzyme that is crucial for gluconeogenesis (the synthesis of glucose from non-carbohydrate sources, like fats and protein), even though the precise gluconeogenesis pathway is unknown for S. solfataricus. Likewise, they detected the enzyme carbon monoxide dehydrogenase, which implies that S. solfataricus might be able to use carbon monoxide as a source of energy, even though scientists previously thought it was purely aerobic.

These findings demonstrate how much more there is to discover about this extremophile's proteome and metabolic pathways, and Dratz and his team are currently developing new variations of 2-DE to help study them. "We continue to use S. solfataricus as a test system to assess the development of new differential fluorescence detection technology in 2-DE," he told SeparationsNOW. "[This will] provide increased detection sensitivity...and promises improved recovery of difficult proteins."

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