Speeding up evolution to create useful proteins wins the chemistry Nobel

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Techniques that put natural evolution on fast-forward to build new proteins in the lab have earned three scientists this year’s Nobel Prize in chemistry.

Frances Arnold of Caltech won for her method of creating customized enzymes for biofuels, environmentally friendly detergents and other products. She becomes the fifth woman to win the Nobel Prize in chemistry since it was first awarded in 1901. Gregory Winter of the University of Cambridge and George Smith of the University of Missouri in Columbia were recognized for their development and use of a technique called phage display. This molecule-manufacturing process can generate biomolecules for new drugs.

The trio will share the 9-million-Swedish-kronor prize (about $1 million), with Arnold getting half and Winter and Smith splitting the other half.

“Wow, well-deserved!” says Paul Dalby, a biochemical engineer at University College London. “Protein engineering as a field is absolutely founded upon their work.”

In the 1990s, Arnold wanted to make an enzyme that would break down a milk protein called casein in an organic liquid, rather than in water. Instead of trying to manually sculpt the chemical building blocks of that enzyme, subtilisin E, to give it the right properties, she opted for a more hands-off approach.

<img src="data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAAIAAAABCAIAAAB7QOjdAAAAGXRFWHRTb2Z0d2FyZQBBZG9iZSBJbWFnZVJlYWR5ccllPAAAAyZpVFh0WE1MOmNvbS5hZG9iZS54bXAAAAAAADw/eHBhY2tldCBiZWdpbj0i77u/IiBpZD0iVzVNME1wQ2VoaUh6cmVTek5UY3prYzlkIj8+IDx4OnhtcG1ldGEgeG1sbnM6eD0iYWRvYmU6bnM6bWV0YS8iIHg6eG1wdGs9IkFkb2JlIFhNUCBDb3JlIDUuNi1jMTM4IDc5LjE1OTgyNCwgMjAxNi8wOS8xNC0wMTowOTowMSAgICAgICAgIj4gPHJkZjpSREYgeG1sbnM6cmRmPSJodHRwOi8vd3d3LnczLm9yZy8xOTk5LzAyLzIyLXJkZi1zeW50YXgtbnMjIj4gPHJkZjpEZXNjcmlwdGlvbiByZGY6YWJvdXQ9IiIgeG1sbnM6eG1wPSJodHRwOi8vbnMuYWRvYmUuY29tL3hhcC8xLjAvIiB4bWxuczp4bXBNTT0iaHR0cDovL25zLmFkb2JlLmNvbS94YXAvMS4wL21tLyIgeG1sbnM6c3RSZWY9Imh0dHA6Ly9ucy5hZG9iZS5jb20veGFwLzEuMC9zVHlwZS9SZXNvdXJjZVJlZiMiIHhtcDpDcmVhdG9yVG9vbD0iQWRvYmUgUGhvdG9zaG9wIENDIDIwMTcgKFdpbmRvd3MpIiB4bXBNTTpJbnN0YW5jZUlEPSJ4bXAuaWlkOkQ0OTU4Nzk4RTcwMDExRTc4REVDOUM3QzgxMzY3QzExIiB4bXBNTTpEb2N1bWVudElEPSJ4bXAuZGlkOkQ0OTU4Nzk5RTcwMDExRTc4REVDOUM3QzgxMzY3QzExIj4gPHhtcE1NOkRlcml2ZWRGcm9tIHN0UmVmOmluc3RhbmNlSUQ9InhtcC5paWQ6RDQ5NTg3OTZFNzAwMTFFNzhERUM5QzdDODEzNjdDMTEiIHN0UmVmOmRvY3VtZW50SUQ9InhtcC5kaWQ6RDQ5NTg3OTdFNzAwMTFFNzhERUM5QzdDODEzNjdDMTEiLz4gPC9yZGY6RGVzY3JpcHRpb24+IDwvcmRmOlJERj4gPC94OnhtcG1ldGE+IDw/eHBhY2tldCBlbmQ9InIiPz5Sc9lyAAAAEklEQVR42mJ89+4dAwMDQIABAA4AAsyHwrk2AAAAAElFTkSuQmCC" data-echo="" alt class="caption" title="ENZYME EVOLUTION The enzyme subtilisin E, shown here from front (left) and rear (right), is found in laundry detergents and helps break down peptides. Directed evolution in Frances Arnold’s lab resulted in eight amino acid substitutions (blue) that increased the enzyme’s heat stability by 18 degrees Celsius. Peptides attached to the enzyme are shown in gray. ~~ F. Arnold”><img src="" alt class="caption" title="ENZYME EVOLUTION The enzyme subtilisin E, shown here from front (left) and rear (right), is found in laundry detergents and helps break down peptides. Directed evolution in Frances Arnold’s lab resulted in eight amino acid substitutions (blue) that increased the enzyme’s heat stability by 18 degrees Celsius. Peptides attached to the enzyme are shown in gray. ~~ F. Arnold”>

Arnold’s insight “was to recognize that the most amazing molecules in the world weren’t created by chemists, but rather by the biological world,” says Jesse Bloom, a microbiologist at the Fred Hutchinson Cancer Research Center in Seattle. “Biology didn’t make these chemicals using the methods we might learn in an organic chemistry class — rather, it worked by evolution.”

Arnold first made many copies of the original enzyme, each with a different set of genetic mutations. She then inserted the genes for those enzymes into bacteria. These bacteria served as living factories, churning out many copies of each enzyme variant. Arnold picked out the version that did the best job breaking down casein in an organic solvent and repeated the mutation process, starting with that enzyme.

Catalytic action

Frances Arnold’s lab put the enzyme subtilisin E through directed evolution to make it more active in an organic liquid. The changes to create the first variant are shown here in yellow; the molecule was then further evolved to include the additional mutations shown in blue. Molecules and ions bound to the enzyme are pictured in grey.

<img src="data:image/png;base64,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" data-echo="" alt class="caption" title="~~ L. You and F.H. Arnold/Protein Engineering, Design and Selection 1996″><img src="" alt class="caption" title="~~ L. You and F.H. Arnold/Protein Engineering, Design and Selection 1996″>

After several rounds of mutating genes and choosing only the best enzyme to advance to the next round, Arnold was left with an extremely efficient custom-made enzyme. This enzyme-tailoring technique, known as directed evolution, allows scientists to fine-tune proteins the same way nature does, but thousands of times faster. Researchers have used directed evolution to create enzymes that jump-start chemical reactions for creating new drugs and eco-friendly biofuels. Arnold’s lab has also used directed evolution to create enzymes that help forge chemical connections not found in nature, such as bonds between carbon and silicon atoms (SN: 12/24/16, p. 11).

The other half of the Nobel Prize honors work on a molecule-making procedure known as phage display. The primary tool for this process is a type of virus known as a bacteriophage — a simple microbe made of genetic material enclosed in a protein package.

In the 1980s, Smith got the idea to insert the genes responsible for producing various specific proteins into bacteriophages’ genetic code, creating phages that bore these proteins on their surface. Smith’s original motivation was identifying which genes created which proteins. By fishing around a bacteriophage soup with a molecule known to bind to a certain protein, Smith surmised, a researcher could pick out only the phage armed with that particular protein and discover which gene allowed the phage to create it.

In 1985, Smith manipulated bacteriophage DNA to create a phage carrying a piece of protein, called a peptide, that wouldn’t naturally occur on its surface. He then used a peptide-binding molecule to pick this bacteriophage out of the crowd. Smith’s method of meddling with bacteriophage DNA is the foundation of phage display. Since his seminal work producing a peptide-bearing phage, other researchers have adopted his method to create phages that harbor other biomolecules, like antibodies.

Our bodies naturally produce hundreds of thousands of different antibodies that are designed to latch on to viruses and bacteria — essentially putting a hit on these invaders for the immune system to destroy. But researchers had long wanted to create in the lab antibodies that work as medications to curb various diseases. In 1990, Winter used the phage display method to create a phage armed with part of an antibody that binds to the molecule phOx. Winter then used phOx to collect the phage with the antibody from a collection of 4 million other phages.

To ensure that he was using phages to farm the best antibodies possible, Winter then adopted a similar method of directed evolution as Arnold. Winter first created a pool of bacteriophages genetically programmed to produce billions of different antibodies. From that group, he could use a target molecule, like phOx, to collect only the antibody-carrying phages that bound to it the best. From those phages, Winter created a new generation of antibody-toting viruses, and once again used the target molecule to pick out only the best of the bunch.

In the 1990s, Winter and his colleagues used this survival-of-the-fittest-phage technique to produce the antibody adalimumab, creating a drug that neutralizes a chemical that incites inflammation in patients with autoimmune diseases. The drug, known as Humira, was cleared to treat rheumatoid arthritis in 2002 and is now also used to treat psoriasis and inflammatory bowel disease (SN: 8/1/09, p. 8).

Packing peptides

George Smith developed a method called phage display that farms for biomolecules. First, the researcher inserts a gene for making a particular molecule (here, a piece of protein called a peptide) into a phage’s genetic code (1). That creates a phage programmed to produce that peptide on its surface (2). Using an antibody that binds to that peptide, the researcher can harvest the peptide-carrying phage from a soup of many phages (3).

Phage display “is an extremely versatile technology,” says Jonathan Lai, a biochemist at the Albert Einstein College of Medicine in New York City, whose lab uses phage display to develop vaccines. Other researchers have used phage display to produce antibodies that help treat the autoimmune disease lupus and fight cancer.

Directed evolution and phage display provide “a great demonstration of how studying fundamental biological questions like the natural process of evolution can lead to great breakthroughs in technology and medicine,” says Jon Lorsch, director of the National Institute of General Medical Sciences in Bethesda. Md.

These techniques could be used to create molecules that we haven’t yet discovered or have never even considered, says Peter Dorhout, president of the American Chemical Society. “It’s an open frontier.”

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