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Survival of the Fittest Molecule Survival of the Fittest Molecule Biochemists harness a novel form of evolution to sculpt new compounds for the fight against dengue fever, cancer and other modern plagues Willem Stemmer and Brett Holland ver billions of years, life has in the proteins encoded by these genes. increased the speed and efficiency of Oevolved into a spectacular diversi- Out of these modified proteins, a small their creation. ty of forms—more than a million fraction worked in a superior or novel species presently exist. For each, the way and gave some advantage to their Biotech Promise source of its uniqueness is the particu- bearers; ultimately, organisms with the Proteins make and maintain life, so lar constellation of proteins found with- mutation left more descendants than they play an understandably central in its cells. Yet in the midst of this di- those without it. In other words, muta- role in biomedical science. Many hu- versity, the similarities between living tion provides the raw material for nat- man maladies come from disease things are profound. For example, al- ural selection. genes that cause specific protein though the fruit fly genome encodes Evolution also works over shorter changes, including hemophilia, mus- about 14,000 different proteins, and hu- timescales. Anatomically modern hu- cular dystrophy and sickle cell anemia. mans have two to three times that mans are probably less than 170,000 While some of these altered-protein number, many proteins are still recog- years old (see “We Are All Africans,” diseases remain difficult to correct, oth- nizably similar in sequence and task, page 496), yet the genetic diversity ers can be treated. In the case of classic reflecting their common ancestry. In among living Homo sapiens runs to sev- hemophilia, replacing the blood- fact, when scientists have put human eral million mutations, also known as clotting factor VIII protein that is miss- disease genes into flies, they often cause polymorphisms, which form the basis for ing or reduced in people with the dis- the same symptoms in the insects as a vast number of combinations, each ease can lessen the chronic bleeding they do in people. Furthermore, addi- with a potentially different set of prop- problems. The original source for this tion of a normal human gene can some- erties. Because preserving a low muta- purified protein was plasma from hu- times compensate for the deletion of tion rate is important for complex or- man blood, but in 1984 the normal ver- the same gene from the fly. ganisms, the principal source of sion of the factor VIII gene was insert- The differences that do exist be- functional genetic diversity is recombi- ed into a microbial host. These trans- tween equivalent genes in flies and hu- nation between sister chromosomes, a formed bacteria were designed to pro- mans are the result of 900 million or so process that creates new combinations duce massive quantities of the human years of DNA mutations—the amount from existing point mutations. These factor VIII protein, and in 1990 a large of time that has passed since the diver- rearrangements are not necessarily clinical trial demonstrated the success gence of arthropods (including fruit beneficial—many are neutral or harm- of the new drug. The recombinant pro- flies) and chordates (including human ful—but the process is endlessly itera- tein was safer as well as more efficient beings). Some of these mutations also tive, creating in each generation novel to produce: Bioengineered factor VIII changed the sequence of amino acids combinations of mostly old mutations. freed patients from the risks associated Indeed, recombination followed by with blood borne diseases such as HIV Willem “Pim” Stemmer invented DNA breeding natural selection is the foremost mech- and hepatitis. and co-founded Maxygen Incorporated in 1997 to anism of organic evolution. For all its promise, the biotechnology develop the technology. Currently, Maxygen and Now biomolecular engineers seek- revolution is still relatively young. The its subsidiaries, Codexis and Verdia, are focused on ing to craft proteins that will perform first therapeutic use of genetically engi- applying this technique to pharmaceuticals, vac- certain functions have co-opted this neered human proteins was only 25 cines, chemicals and agriculture, and Pim has co- evolutionary strategy. Pharmacologists years ago, after recombinant insulin founded a new company called Avidia Research In- and industrial biochemists are patently was produced in bacteria. Such thera- stitute to focus on antibody-like molecules. Brett interested in tailoring proteins to indi- pies have exploded since then, aided by Holland is an assistant professor of biology at Cali- vidual needs, and developing the tools the increasing sophistication of tools for fornia State University in Sacramento, where he teaches evolution and genetics. His research inter- to create these molecular thorough- manipulating DNA. The main advance ests include sexual selection and sexually antago- breds is crucial. In this article, we dis- was the brilliantly simple use of a com- nistic coevolution. Dr. Stemmer’s address is cuss the importance of custom-built bination of two well-studied classes of Avidia, 2450 Bayshore Drive, Mountain View, CA proteins and describe some of our own bacterial enzymes: restriction endonu- 94043. Internet: [email protected] breakthroughs that have dramatically cleases and ligases. These enzymes cut © 2003 Sigma Xi, The Scientific Research Society. Reproduction with permission only. Contact [email protected]. 526 American Scientist, Volume 91 Figure 1. A strain of the bacteria Streptomyces ambofaciens produces the antibiotic polyketide spiramycin, seen as beads of clear liquid on the surface of each blue colony. In this article, the authors discuss their development of strategies to optimize the function of proteins and entire bacterial genomes by mimicking in vitro the natural process of DNA recombination—an approach that they term “DNA breeding.” (Photograph courtesy of Tobias Kieser, John Innes Centre, Norwich, U.K.) and rejoin, respectively, pieces of DNA Improving Nature’s Design for the optimization and redesign of in a sequence-specific, origin-indepen- In light of this inherent limitation, an im- proteins. They are generally known as dent manner. This allowed molecular portant goal of biotechnology is to re-op- rational design and directed evolution. biologists to create hybrid DNA mole- timize molecules to preserve or enhance Rational design, also known as com- cules from different species, meaning their function. For example, some indus- puter modeling, attempts to modify or that genes could be copied, or cloned, trial enzymes need to be modified for ex- create molecules for specific applica- endlessly by putting them into fast-di- pression at extremely high levels, yet re- tions by predicting which amino acid viding bacteria. Likewise, the proteins tain their function under harsh physical sequence will produce a protein with encoded by DNA could be made in conditions. In the pharmaceutical indus- the desired properties. Using a combi- quantities that were impossible to get try some human proteins may require a nation of x-ray crystallography, three- from the original sources. different receptor-binding property or cir- dimensional structure determination Modern molecular technologies now culatory half-life to survive therapeutic and computer-simulated amino acid provide unprecedented access to the administration. Another example comes interactions, biochemists can often pre- enormous diversity of natural proteins. from our own lab, where we are optimiz- dict which amino acid substitutions are However, these proteins tend to be ing a viral vaccine to induce a strong and the best candidates to elicit the desired very dependent on the cellular context broadly protective immune response. change in a protein. in which they evolved. When they are This is a challenge because viruses have Unfortunately, the task of accurately removed from the original cellular mi- evolved specifically to evade the immune modeling protein function is Her- lieu and produced and assayed in a systems of their hosts. culean—there are a staggering number different environment, the proteins are At present two different but comple- of interdependent variables that influ- apt to perform poorly or not at all. mentary strategies are being pursued ence protein function. Cells go through © 2003 Sigma Xi, The Scientific Research Society. Reproduction with permission only. Contact [email protected]. www.americanscientist.org 2003 November–December 527 many steps between DNA and an ac- tive protein—including but not limit- ed to RNA and peptide synthesis, post- translational modification, subcellular targeting and intermolecular bind- ing—and each step is regulated by multiple mechanisms. Protein folding and stability alone are sensitive to dozens, if not hundreds, of internal and external factors, and the consider- ation of any additional properties, such as activity in the presence of organic solvents, complicates matters further. Even without calculating such interac- lker Steger/Science Photo Library Vo tions between molecules, modeling the forces exerted by amino acids within Figure 2. Insulin was the first recombinant protein produced by genetically engineered bacte- ria for therapeutic use in humans beings. This pseudocolored transmission electron micro- the protein is an enormous undertak- graph shows Escherichia coli bacteria carrying the human insulin gene; sites
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