BRCA2, purified, reveals some of its secrets.

according to Gordon Mills, M.D., Ph.D., chair of molecular oncology at the M. D. Anderson Cancer Center in Houston. “I’d describe it as a technical tour de force,” said Mills, who was not involved in the work. “It refi nes our understanding of the mechanism by which BRCA2 coordinates DNA repair, and it could have a massive impact on developing new ways to go after abnormalities in both BRCA1 and BRCA2.” The results were published online in Nature and Nature Structural Biology in August. BRCA2 ranks among the largest human proteins, with 3,418 amino acids. Scientists struggled for 15 years to purify the giant molecule, which tends to break into fragments almost as soon as it’s transcribed in the laboratory. To get around this problem, researchers had to affi x protein tags to BRCA2’s front and back ends, which held the molecule in place during extraction from cell-based expression systems. Remarkably, each research team purifi ed BRCA2 around the same time, though each used different tags and different systems. Stephen C. Kowalczykowski, Ph.D. , a biochemist at the University of California, Davis, who led the team that published in Nature , said that hitting on the right combination of tags and expression systems took considerable trial and error. Neither bacterial nor insect cell lines possessed the protein mix that human BRCA2 relies on to fold correctly as it emerges from the genome, he said. Kowalczykowski’s postdoctoral student, Ryan Jensen, Ph.D., ultimately succeeded by using human cells and tags made of maltose binding protein, which attach preferentially to amylose resins used in chromatography columns. Like magnets on a refrigerator door, these proteins hold BRCA fast during the extraction, trapping the molecule while other proteins in the cell slip by. Wolf-Dietrich Heyer, Ph.D., also a biochemist at UC Davis, and lead author on one of the Nature Structural Biology reports, successfully purifi ed BRCA2 in yeast, using a glutathione S -transferase tag on the protein’s N terminus (front end) and a histidine affi nity tag on its C terminus (back end). “We used yeast because we’ve got a lot of experience with it,” Heyer said. “We had a lot of tricks we could use to express the protein and also to preserve it.” Finally, Stephen West, Ph.D., a geneticist at Cancer Research UK in London, and colleagues, who also published in Nature Structural Biology , purifi ed BRCA2 in cultured human HeLa cells made in a bacterial construct. While acknowledging the purifi cation’s importance to science, Kowalczykowski also describes the achievement as more of a means to an end. “The more important story is what we can do with the full-length validated protein in hand,” he said. “Now we can run detailed mechanistic and molecular studies to explore what BRCA2 does.” “The more important story is what we can do with the full-length validated protein in hand.”