Nobel Prize in Chemistry 2018: Directed Evolution and Phage Display

This note covers the Nobel Prize in Chemistry 2018: who won it, what directed evolution of enzymes and phage display of peptides and antibodies mean, how Frances Arnold, George Smith and Gregory Winter carried out their work, how the discovery unfolded, why it matters for medicine and industry, and quick facts for exams.

What was the Nobel Prize in Chemistry 2018 awarded for?

The Nobel Prize in Chemistry 2018 was split between two separate pieces of work. One half went to Frances H. Arnold with the citation "for the directed evolution of enzymes". The other half was shared jointly by George P.

Smith and Sir Gregory P. Winter with the citation "for the phage display of peptides and antibodies".

In plain words, all three laureates found ways to copy a trick that nature itself uses. Living things change their proteins over many generations through random genetic change followed by selection of the most useful variants, a process called evolution.

Arnold used this trick inside test tubes and bacteria to improve enzymes (proteins that speed up chemical reactions). Smith and Winter used a similar trick, built around a bacteria-infecting virus, to improve antibodies and other proteins used in medicine.

The official body that decides this prize is the Royal Swedish Academy of Sciences, and the award is formally called the Nobel Prize in Chemistry.

Who are the laureates?

Frances H. Arnold

Frances H. Arnold was born on 25 July 1956 in Pittsburgh, PA, USA. At the time of the award she worked at the California Institute of Technology (Caltech), Pasadena, USA, where she held the title of Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry.

She received one half of the prize. She trained in mechanical and aerospace engineering at Princeton, then completed a doctorate in chemical engineering at the University of California, Berkeley, in 1985.

In 1993 she carried out the first directed evolution of an enzyme, and and in 2005 she set up a company to make renewable fuels using evolved enzymes.

George P. Smith

George P. Smith was born on 10 March 1941 in Norwalk, CT, USA. At the time of the award he was at the University of Missouri, Columbia, USA. He received one quarter of the prize.

He studied at Haverford College and then at Harvard University, where his doctorate, completed in 1970, was in bacteriology and immunology.

While at Duke University in 1983 to 1984 he began the work that earned him the prize: in 1985 he developed phage display, a method of using a bacteriophage (a virus that infects bacteria) to link an unknown protein to its gene.

Sir Gregory P. Winter

Sir Gregory P. Winter was born on 14 April 1951 in Leicester, United Kingdom. At the time of the award he was at the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. He received one quarter of the prize.

He grew up in Ghana, read natural sciences at Cambridge, and completed his doctorate in 1977 while working at the MRC Laboratory of Molecular Biology (nobelprize.org's press release instead gives 1976). From 1990 he adapted George Smith's phage display method to evolve antibodies, work that led to new antibody-based medicines.

What problem were the laureates trying to solve?

Since the first life appeared on Earth around 3.7 billion years ago, evolution has filled almost every environment on the planet with organisms suited to it, from hot springs to deep oceans to dry deserts.

The Nobel Committee said this huge variety exists because evolution has solved countless chemical problems using proteins as its tools.

Chemists, however, had traditionally tried to design new molecules by reasoning out their structure logically.

By the late 1980s, Frances Arnold found this approach very hard for enzymes because an enzyme can contain thousands of linked amino acids folded into complicated three-dimensional shapes, and working out by logic how to change that shape to get a new property was, in her own words quoted by the Academy, a "somewhat arrogant approach".

She decided instead to copy nature's own method: change genes randomly and then select the best result.

Separately, in the early 1980s molecular biologists faced a different problem. The human genome held the gene for every protein in the body, but finding the specific gene behind a specific protein was described as being as hard as finding a needle in a haystack.

George Smith wanted a quicker way to connect a known protein to its unknown gene, and this led him to bacteriophages, the simple viruses that infect bacteria.

At the same time, drug developers struggled to make safe antibody medicines. Antibodies made by injecting mice could be toxic to the animals, failed to form in some cases, and were often recognised as foreign by a human patient's immune system, causing side effects.

Gregory Winter wanted antibody drugs built on human antibody genes instead of mouse ones, and found his answer in Smith's phage display method.

How does directed evolution of enzymes work?

An enzyme is a protein that speeds up, or catalyses, a chemical reaction. Frances Arnold's method of directed evolution repeats the natural cycle of mutation and selection inside the laboratory, instead of trying to redesign an enzyme by pure reasoning.

1. Start with a working enzyme, for example the enzyme subtilisin, and introduce random changes (mutations) into the gene that codes for it.
2. Insert the mutated genes into bacteria, so that each bacterium produces one mutated version of the enzyme, creating thousands of different variants.
3. Test, or select, which variant performs the desired job best; Arnold tested which subtilisin variant broke down a milk protein, casein, most effectively in a solvent called dimethylformamide (DMF).
4. Take the best-performing variant and repeat the whole cycle of random mutation and selection on it, generation after generation.

In Arnold's own early experiment, by the third generation she found a subtilisin variant that performed 256 times better in DMF than the original enzyme, carrying a combination of ten different mutations that nobody could have predicted in advance.

A separate Dutch researcher, Willem Stemmer, added a further idea in 1994 called DNA shuffling, cutting up different gene versions and reassembling the pieces, mimicking the mixing of genes seen in natural mating.

Draw a circular arrow diagram with four stages labelled mutate, express in bacteria, select the best performer, and repeat; show the enzyme's effectiveness rising with each loop around the circle.

Arnold's laboratory later produced enzymes that catalyse reactions not found in nature at all, and developed enzymes that convert simple sugars into isobutanol, a substance usable for biofuels and greener plastics.

How does phage display of peptides and antibodies work?

A bacteriophage (often shortened to phage) is a simple virus that infects bacteria: a small piece of genetic material wrapped in protective proteins.

George Smith's insight was that if a researcher attached an unknown gene fragment to the gene for one of the phage's own coat proteins, the protein made from that unknown gene would end up displayed on the surface of the new phage.

1. Join many different unknown gene fragments to the gene for a phage coat protein, creating a large mixed population of phages.
2. Let the phages reproduce inside bacteria, so each new phage carries a different protein or peptide fragment on its surface.
3. Use an antibody that is known to bind a particular target protein as a kind of fishing hook, and dip it into the mixed soup of phages.
4. Pull out only the phages whose surface protein was caught by the hook; because each phage still carries its own genetic blueprint, the previously unknown gene for that protein is now identified.

In 1985, Smith proved this worked by displaying part of a protein (a peptide) on a phage and fishing it out of a mixed soup using a matching antibody.

Gregory Winter then turned the idea around: instead of searching for unknown genes, he displayed antibody fragments themselves on phages and used phage display to evolve better antibodies.

In 1990 he showed that an antibody's binding arm, joined to a phage coat protein gene, ended up correctly displayed on the phage surface, and he used a small molecule called phOx as bait to pull his engineered phage out of a soup of four million others.

Draw a phage particle with a coat protein carrying a displayed peptide or antibody fragment on its surface, with an arrow showing an antibody-coated "hook" catching only the matching phage out of a large mixed group.

By repeating rounds of random change and selection on the displayed antibody library, Winter produced antibodies that bound their targets ever more tightly, the same mutate-and-select logic that Arnold used for enzymes, applied instead to antibody proteins.

What medicines and products has this work produced?

The clearest result of Winter's antibody work is adalimumab, the first pharmaceutical based entirely on a human antibody obtained through phage display. It works by neutralising a protein called TNF-alpha, which drives inflammation in several autoimmune diseases.

It was approved in 2002 for rheumatoid arthritis and has since also been used for types of psoriasis and inflammatory bowel disease.

Phage display has since produced further antibody medicines, including ones that neutralise toxins, counteract autoimmune diseases, and in some cases help cure metastatic cancer, described by the Nobel Committee as a historic breakthrough in cancer care.

Arnold's directed-evolution enzymes, meanwhile, are used in more environmentally friendly manufacturing of substances such as pharmaceuticals, and in producing renewable fuels for a greener transport sector.

How did the discovery unfold?

The table below traces the main steps named in the Nobel sources, from Smith's first phage display experiment to the award itself.

Why does this discovery matter?

The Nobel Committee said the laureates had "harnessed the power of evolution" and used it for the greatest benefit of humankind. Directed evolution lets chemists design enzymes without needing to fully understand their complicated internal structure in advance, simply by letting mutation and selection do the work across repeated generations.

In industry, Arnold's evolved enzymes have reduced the need for the strong solvents, heavy metals and corrosive acids used in older chemical manufacturing, cutting environmental impact while making processes such as pharmaceutical production more efficient. Her work on turning sugars into isobutanol points towards greener fuels for cars and aeroplanes.

In medicine, phage display solved the long-standing problem of making antibody drugs that the human immune system does not reject, because the antibodies can now be built from human genetic sequences rather than from mice.

This opened the way to treatments for autoimmune disease, toxin exposure and some cancers. The press release described this as being in "the early days of directed evolution's revolution", suggesting the Committee expected many further applications to follow.

How does this connect to what you study?

This prize links directly to the genetics ideas of mutation, variation and selection taught in school biology, and to basic chemistry ideas about enzymes as catalysts and proteins as chains of amino acids. The central idea that small random changes in genes can be tested and the best result kept for the next round is exactly the mechanism behind natural selection, so this prize is a useful real-world case study for that topic.

Phage display also connects to the idea of a virus as a simple genetic parcel, something students meet when studying microorganisms and the immune system. A bacteriophage is built from genetic material wrapped in protective proteins, and understanding how it injects its genes into a bacterium and hijacks the bacterium's machinery to make copies of itself helps explain how scientists turned this natural process into a laboratory tool.

The antibody side of the story touches the immune system topic directly: antibodies are Y-shaped proteins that bind very precisely to a single target among thousands of others, which is the same idea taught when explaining how the body recognises and fights infection.

Seeing how laboratory evolution produced a real medicine, adalimumab, shows how fundamental biology and chemistry concepts, genes, proteins, mutation and selection, translate into working treatments used in hospitals today, which is a useful link between classroom theory and applied science.

Quick facts for exams

The Nobel Prize in Chemistry 2018 was announced on 3 October 2018 by the Royal Swedish Academy of Sciences. It was divided between Frances H.

Arnold of Caltech, USA, who received one half for the directed evolution of enzymes, and George P. Smith of the University of Missouri, USA, together with Sir Gregory P.

Winter of the MRC Laboratory of Molecular Biology, UK, who shared the other half for the phage display of peptides and antibodies. Arnold evolved enzymes in the laboratory using repeated rounds of random mutation and selection;

Smith invented phage display, a method linking an unknown protein to its gene using a bacteriophage; Winter applied phage display to evolve human antibodies, leading to the drug adalimumab. The total prize amount that year was 9,000,000 Swedish kronor.

Note: Source. The prize facts in this note are from the Nobel Prize's official site, nobelprize.org.

Glossary

• Directed evolution — a laboratory method of repeatedly mutating a gene and selecting the best-performing protein, copying natural evolution to improve enzymes or antibodies.
• Enzyme — a protein that speeds up (catalyses) a chemical reaction without being used up itself.
• Catalyst — a substance that speeds up a chemical reaction.
• Mutation — a random change in the genetic code of an organism or protein.
• Bacteriophage (phage) — a simple virus that infects bacteria, consisting of genetic material wrapped in protective proteins.
• Phage display — a method of attaching a gene for an unknown or engineered protein to a phage's coat protein gene so the protein appears on the phage surface.
• Antibody — a Y-shaped protein made by the immune system that binds very specifically to a target molecule.
• Peptide — a short chain of amino acids, smaller than a full protein.
• Amino acid — one of the building-block molecules that are linked together to form proteins.
• DNA shuffling — a recombination method that cuts gene fragments and reassembles them into new mixed versions, speeding up directed evolution.
• Adalimumab — the first drug based on a fully human antibody developed through phage display, used for rheumatoid arthritis and related diseases.
• TNF-alpha — a protein in the body that drives inflammation in several autoimmune diseases, and which adalimumab neutralises.
• Autoimmune disease — a condition in which the immune system mistakenly attacks the body's own tissues.
• Isobutanol — an energy-rich chemical that can be produced from sugars using evolved enzymes, used for biofuels and plastics.
• Selection — the step in evolution, natural or directed, where the best-performing variant is chosen to continue to the next generation.

Common errors and misconceptions

• Misconception: Directed evolution means designing a new enzyme on a computer. Correct: it relies on repeated rounds of random mutation and practical selection, not full rational design.
• Misconception: Phage display is dangerous because it uses a virus that can infect people. Correct: bacteriophages infect only bacteria, and in this method they are simply used as display and copying tools.
• Misconception: George Smith invented adalimumab. Correct: Smith invented phage display itself; Gregory Winter later applied it to evolve the antibody behind adalimumab.
• Misconception: The 2018 Chemistry Prize is about editing the human genome. Correct: it is about evolving enzymes and antibodies in the laboratory, not altering human genes.
• Misconception: A single round of mutation and selection is enough for a big improvement. Correct: Arnold's own experiment needed three generations of mutation and selection to reach a 256-fold improvement.
• Misconception: Antibody drugs are always made from mice. Correct: phage display allowed Winter to build antibodies from human genetic sequences, avoiding problems caused by mouse-derived antibodies.
• Misconception: All three laureates received an equal one-third share. Correct: Arnold received one half of the prize, while Smith and Winter shared the other half equally.

Exam-style questions with model answers

Q1. State the official citation for the half of the Nobel Prize in Chemistry 2018 awarded to Frances Arnold. [2 marks]

1. The citation was "for the directed evolution of enzymes", honouring her method of improving enzymes through repeated mutation and selection in the laboratory.

Q2. Name the two laureates who shared one half of the Nobel Prize in Chemistry 2018 for phage display. [2 marks]

1. George P. Smith and Sir Gregory P. Winter shared one half of the prize for the phage display of peptides and antibodies.

Q3. Explain what directed evolution of enzymes means and why it is useful. [4 marks]

1. Directed evolution is a laboratory method that copies the logic of natural evolution: a gene is mutated randomly, the resulting protein variants are tested, and the best performer is carried forward into a further round of mutation and selection. It is useful because enzymes are extremely complex molecules built from thousands of linked amino acids folded into intricate shapes, making it very hard to redesign them by pure reasoning. By letting chance and selection do the work across several generations, Frances Arnold was able to create enzymes with new or greatly improved abilities, such as working efficiently in an organic solvent, without needing to understand every detail of their structure in advance.

Q4. Describe the steps Frances Arnold used in evolving the enzyme subtilisin. [4 marks]

1. She introduced random mutations into the gene for subtilisin and put the mutated genes into bacteria, producing thousands of different enzyme variants. She then selected the variant that broke down the milk protein casein most effectively in a solution containing dimethylformamide (DMF), an organic solvent the enzyme does not normally work well in. She repeated this cycle of mutation and selection on the chosen variant for further generations. By the third generation she obtained a variant with ten combined mutations that performed 256 times better in DMF compared with the original enzyme.

Q5. Discuss how phage display works and how Gregory Winter used it to develop a human-antibody medicine. [6 marks]

1. Phage display, invented by George Smith in 1985, uses a bacteriophage, a virus that infects bacteria, as a linking tool between a protein and its gene. An unknown or engineered gene fragment is joined to the gene for one of the phage's own coat proteins, so that when new phages are produced, the protein from that fragment appears displayed on the phage's surface. Researchers can then use a matching antibody as a kind of fishing hook to pull the correct phage out of a large mixed population, which also recovers the gene behind the displayed protein. Gregory Winter adapted this idea for antibodies themselves: in 1990 he joined the gene for an antibody's binding arm to a phage coat protein gene and showed the antibody fragment appeared correctly on the phage surface, fishing his engineered phage out of a soup of four million others using a bait molecule called phOx. By building large libraries of phage-displayed antibody fragments and repeating rounds of mutation and selection, Winter evolved antibodies with stronger and more specific binding. This led to adalimumab, the first drug based entirely on a human antibody, approved in 2002 for rheumatoid arthritis and later used for psoriasis and inflammatory bowel diseases, because it neutralises the inflammation-driving protein TNF-alpha.

Q6. Give one example of a disease treated using an antibody developed through phage display. [2 marks]

1. Rheumatoid arthritis is treated using adalimumab, an antibody medicine developed through phage display that neutralises the inflammation-driving protein TNF-alpha.

Q7. Explain what a bacteriophage is and how George Smith used it as a tool. [4 marks]

1. A bacteriophage is a simple virus that infects bacteria, made of a small piece of genetic material wrapped in protective coat proteins. George Smith realised that if an unknown gene fragment was joined to the gene for one of these coat proteins, the protein it coded for would end up displayed on the surface of newly produced phages. He could then use a known antibody as bait to fish out, from a mixed soup of phages, the one carrying the matching protein on its surface, which also revealed the previously unknown gene. In 1985 he proved this worked with a peptide displayed on a phage surface.

Q8. Discuss why the Nobel Committee said the three laureates had harnessed the power of evolution. [6 marks]

1. The Royal Swedish Academy of Sciences explained that evolution, operating over billions of years through random genetic change and selection, has produced an enormous diversity of life because it has solved countless chemical problems through proteins. The Committee said the 2018 laureates had taken control of this same process and used it deliberately for human benefit. Frances Arnold applied mutation and selection inside the laboratory to evolve enzymes, producing catalysts for greener chemical manufacturing and for making renewable fuels such as isobutanol. George Smith and Gregory Winter applied the same principle of genetic change and selection to proteins displayed on bacteriophages, with Winter specifically evolving antibodies rather than relying on unpredictable natural immune responses in animals. The Committee stated that the resulting enzymes and antibodies bring great benefit to humankind, for example enzymes used to manufacture pharmaceuticals and biofuels, and antibodies such as adalimumab that treat autoimmune disease and, in some cases, cure metastatic cancer, describing this as being in the early days of a wider revolution.

Key takeaways

• The Nobel Prize in Chemistry 2018 honoured directed evolution of enzymes and phage display of peptides and antibodies.
• Frances Arnold received one half of the prize for directing evolution inside the laboratory to improve enzymes.
• George Smith received one quarter for inventing phage display in 1985, using a bacteriophage to link a protein to its gene.
• Gregory Winter received one quarter for applying phage display to evolve human antibodies for medicine.
• Directed evolution works through repeated cycles of random mutation and selection, copying natural evolution.
• Phage display displays a protein on a virus surface so it can be fished out using a matching antibody.
• Adalimumab, approved in 2002, was the first drug based on a fully human antibody made using this method.
• The prize amount for 2018 was 9,000,000 Swedish kronor, announced on 3 October 2018.

Test yourself

What citation did Frances Arnold receive?

Frances Arnold received the citation "for the directed evolution of enzymes" for her half of the 2018 prize.

Where was George Smith working when he began the research that led to his Nobel Prize?

George Smith began his Nobel-awarded work at Duke University in 1983 to 1984, before returning to the University of Missouri.

What is a bacteriophage?

A bacteriophage is a simple virus made of genetic material wrapped in protective proteins that infects bacteria, not human cells.

What medicine resulted from Gregory Winter's phage display research, and what disease does it treat?

Winter's research led to adalimumab, approved in 2002 for treating rheumatoid arthritis and later used for psoriasis and inflammatory bowel diseases.

How many rounds of mutation and selection did Arnold need to get a 256-fold improvement in subtilisin?

Frances Arnold reached a 256-fold improvement in subtilisin's performance after three generations of mutation and selection.

How was the Nobel Prize in Chemistry 2018 split between the three laureates?

Frances Arnold received one half of the prize, while George Smith and Sir Gregory P. Winter shared the other half equally.

What problem with mouse-derived antibodies did phage display help to solve?

Phage display let researchers build antibodies from human genetic sequences, avoiding the toxicity and immune rejection seen with mouse-derived antibodies.