UW, Japan collaboration could help chart the protein universe

The billions of proteins that compose life on Earth remain one of the truly uncharted territories in the biological universe, due mainly to the slow and arduous techniques their exploration requires.

Now, a research partnership between UW–Madison and a Japanese university and company aims to develop a technology that may allow scientists to map the shapes and structures of proteins more easily than ever before. The advance promises to help unlock the inner workings of hundreds or even thousands of proteins, according to UW–Madison biochemistry professor John Markley, leading to a better understanding of protein-based diseases, and providing fundamental new information about the building blocks of all living beings, from bacteria to plants to people.

An agreement signed this week by the UW–Madison’s Center for Eukaryotic Structural Genomics (CESG), the university’s patent management agency the Wisconsin Alumni Research Foundation (WARF), Ehime University in Matsuyama, Japan, and the Japanese biotechnology company Cell-Free Sciences of Yokohama, formalizes an ongoing collaboration between these groups to refine a powerful new system, created in Japan, for making the large quantities of purified protein that biochemists need to solve protein structures.

Under the agreement, CESG will become the only “beta test” site in the nation for the system, comparing its success rate and cost with conventional methods of producing protein for structural studies.

Spearheading the effort to expand the technology are principal investigator and CESG director Markley and scientist Dmitriy Vinarov. Markley’s CESG pilot project, funded by the National Institutes of Health Protein Structure Initiative (PSI), uses the tools of nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to visualize the twists, folds and shapes that form each protein’s unique structure. If the new production system works as well as the researchers anticipate, it could in some cases make enough protein for NMR analysis virtually overnight.

“Right now, it typically takes a year to solve the structure of just one protein and costs between $100,000 and $200,000,” says Markley. “What our group as well eight other PSI pilot projects around the country have found is that protein production represents the major bottleneck in solving structures more quickly and inexpensively.

“This technology will potentially open that bottleneck,” he says, allowing scientists to map the human “proteome” in the same way they’ve mapped the human genome. Knowing the structure of abnormal proteins, such as those implicated in Alzheimer’s and “mad cow” disease, could lead to new treatments for those disorders. Scientists might also more easily engineer changes into known proteins or protein fragments to create new protein-based pharmaceuticals.

Currently, large amounts of individual proteins are usually produced by engineering a bacterium, such as E. coli, to produce a foreign protein as part of its normal growth. But proteins from plants and humans tend to take on abnormal structures when made by E. coli, rendering them useless for structural analysis. As a result, CESG’s own state-of-the-art E. coli production system currently works with only 11 percent of the proteins the Center targets for structure determination.

To overcome this problem, scientists have turned to “cell-free” systems, such as the one developed in Japan. Studies conducted in Japan and confirmed by Vinarov at UW–Madison suggest the new system could work with up to 60 percent of CESG’s target proteins.

Originally developed by Professor Yaeta Endo of Ehime University and reported in the Proceeding of the National Academy of Sciences in 2000 and 2002, the technology harnesses the protein-producing capacity of extracts made from the tiny plant embryos inside wheat seeds. Although others had previously used the system, Endo greatly increased its efficiency and stability by devising an extract preparation protocol that included careful washing of the embryos to remove natural inhibitors of protein synthesis present inside the seed.

Endo, Ehime University and Cell-Free Sciences – a biotech company launched to commercialize the technology – have subsequently made the advance the basis of a new robotic system that automates roughly three dozen synthesis steps normally performed manually by scientists. As part of the agreement, the robotic system will be shipped to Markley’s lab from Cell-Free Sciences in mid-November. Markley hopes to have it up and running by year’s end.

“We’ve already demonstrated we can use the wheat germ extract by hand to produce enough protein for structural analysis,” says Vinarov. “The automation will now allow us to evaluate this technology as a possible high-throughput method of protein production.”

“Although it still needs validation,” adds Markley. “This system could become the new platform for large-scale, cell-free protein synthesis. We’re very pleased this collaboration is moving forward.”