Coupling protein sequence to function, thousands of variants at a time.

Even with a load of new data, presenting information to lab-mates at a group meeting can be an uncomfortable experience. Worse still is reporting a total lack of progress. But for postdoctoral fellow Doug Fowler, publicly detailing the latest dead ends in his project turned out to be pivotal.

His ambitious project had been dogged by technical problems since its inception a year and a half earlier. "That experience of laying out all these things that weren't working made me realize there was another way," says Fowler, who works in the lab of HHMI investigator Stanley Fields at the University of Washington (UW) in Seattle.

Fowler and Fields wanted to comprehensively survey—on a larger scale than had ever been done—how genetic changes influence protein function. Typical approaches produce one mutant version of a protein at a time—for example, by converting some amino acids in a sequence to alanine. Instead, they wanted to track thousands of versions of a protein that systematically varied in its sequence. The result would be a high-resolution map of how each component of a protein contributes to its function, bringing insight to basic biology and drug design alike.

"As you think of a complex, three-dimensional surface of a protein, every position there has its own unique profile," says Fields, whose research into protein function has spawned new technologies. "But you could never predict it with what we know."

Fowler and Fields can now obtain these unique profiles experimentally, but not with their initial strategy. They had been trying to develop a protein microarray on which hundreds of thousands of proteins would be generated from corresponding spots of DNA. As he mulled over his discouraging lab group meeting, it dawned on Fowler that his main problem with the microarray—linking DNA sequence to protein function—had been solved more than 20 years before with a procedure called phage display.

With phage display, DNA encoding a protein of interest is introduced into phage viruses, which display the protein on their outer shells. With each virus containing a different protein, a large collection of variant proteins can be studied. Screening the viruses for the ability to bind to other molecules retains the phage that bind and washes away those that don't. Repeating this step enriches the phage population for those carrying the few proteins that are the best binders, and DNA recovered from these phage "winners" identifies them.

Image: VSA Partners

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