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 Volker Steger/Science Photo Library 2 American Scientist, Volume 91 528 amino acidchanges canbetestedthatmayimprove proteinfunction. Based onanunderstanding ofthephysicalprinciples thatgoverntheseinteractions, specific dicts theforcesbetweenaminoacids andidentifieskeysitesthatinteractwithatargetmolecule. mensional structureoftheprotein isdeducedusingx-raydiffraction. Acomputermodel pre- timize aknownprotein,thestarting geneproductispurifiedandcrystallized,thethree-di- Figure 3.Rationalproteindesign isonestrategyfortailoringproteinstospecificneeds.To op- pression areorange. graph shows ria fortherapeuticuseinhumansbeings.Thispseudocoloredtransmissionelectronmicro- Figure 2.Insulinwasthefirstrecombinantproteinproducedbygeneticallyengineeredbacte- amino acidchanges gene toreflect starting gene starting alter starting ? repeat Escherichia coli new protein e xpress improved function test for bacteria carryingthehumaninsulingene;sitesofproteinex- starting protein starting acids tofindimproved substitute key amino e xpress © 2003SigmaXi, TheScientificResearch Society. Reproduction target bindiing indicates whichaminoacids computer simulation with permission only. [email protected]. interact with target of crystalized protein of crystalized x-ray diffraction protein structure predict protein purify evolution, called The mostpowerfulformofdirected Evolution InaTube by medicine,agriculture andindustry. customized tomeetspecificationsset r selection atthemolecularlevelanddi- physical principles,weharnessnatural proteins bycomputersimulationof modify existingproteins ordesignnew creasing success.Ratherthantryingto lowed thisalternateapproach within- of scientists,includingus,havefol- past decade,amuchsmallernumber heavily onfuture technology. Overthe to rationaldesignthatdoesnotrely so the cell. with thousandsofothermoleculesin can describeaprotein’s interactions limited bythedetailwithwhichwe fruitful inthefuture, butitwillremain that thisapproach maybeincreasingly rise ofmicroprocessor speedsuggests sive computationalpower. Thesteady strained bytheirrequirement formas- models are understandablycon- tion composition. is influencedbychangesinthesolu- 300 aminoacids,andeachinteraction ing—an average-sizepolypeptidehas the protein isanenormousundertak- forces exertedbyaminoacidswithin tions betweenmolecules,modelingthe Even withoutcalculatingsuchinterac- solvents, complicatesmattersfurther. as activityinthepresence oforganic ation ofanyadditionalproperties, such and externalfactors,theconsider- dozens, ifnothundreds, ofinternal and stabilityaloneare sensitiveto multiple mechanisms.Protein folding ing—and eachstepisregulated by targeting andintermolecularbind- translational modification,subcellular ed toRNAandpeptidesynthesis,post- tive protein—including butnotlimit- many stepsbetweenDNAandanac- the wolf the dog14,000yearsago.Starting with that ofearlymanwhodomesticated of desirabletraits.Ourtask resembles spring thathavethebestcombination genetic variantsandselect those off- breed themtocreate adiversepool of the same:Selectpromising parents, plants oranimals.Thestrategiesare has supervisedthereproduction of ing, whichisfamiliartoanyonewho modern derivativeofclassicalbreed- animal buta poor domesticcompan- ect theevolutionofproteins thatare Directed evolutionisanalternative W ith allofthesevariables,computer Canis lupus DNA breeding —a magnificent , isa ion—prehistoric humans selectively function. The method used to screen starting gene bred those individuals with favorable these candidates depends on the func- traits and shaped hundreds of dog tion of the protein. If binding a specific introduce breeds in a relatively short time. Some molecular target, like a receptor or lig- variation with dogs were even bred to perform highly and is critical, then the target can be random point specialized roles, such as sheepherding immobilized on a solid support. A so- mutations in border collies—a prime example of lution containing the pool of new mol- why this technique is powerful. Herd- ecules is passed over the target, and ing behavior is so far removed from molecules that bind it are retained as the wolf’s original behavioral genetic nonbinding proteins wash away. To program that it could never have been cull the strongest-binding proteins, the designed rationally, even with the most immobilized target is washed several sophisticated models and the most times with increasingly stringent solu- powerful computers imaginable. By tions until only a small number of mol- contrast, directed evolution does not ecules remain. Other screening meth- require a priori knowledge of how a ods are used if some catalytic ability is system works. Its great advantage lies sought: Reaction rates can be evaluated in the ability to modify a complex through chemically linked indicator property without knowing every detail molecules that change color as the re- of its mechanism. action proceeds. With this technique, DNA breeding makes use of estab- automated optical assays can screen select best lished biochemical techniques to apply thousands of reactions per hour for the candidates strong selective pressure to molecules color intensity within each sample. for breeding rather than whole animals. And be- Once the population of mutated cause the evolution takes place in test compounds has been winnowed to a tubes rather than in kennels, the entire scant number of contenders, positive process is notably faster. mutations and a few remaining nega- The basic advance of DNA breed- tive mutations from the selected candi- ing is the recombination of diverse ge- dates are refashioned into new combi- netic material into novel and poten- nations. This generates an entirely new tially more productive forms. This but closely related pool of molecules to strategy can be applied at many lev- analyze for the targeted trait. Thus, the cut and els, with the first requirement being iterative process of diversity genera- reform using some reservoir of genetic variation. In tion and selection is repeated until we polymerase chain reaction the simplest form of DNA breeding, achieve the desired quality or combi- which uses only a single gene, the nation of qualities. progeny functional diversity that is normally provided by a natural population DNA Family Breeding needs to be generated in the laborato- Directed evolution through random ry. To do this, thousands of copies of mutation of a single starting sequence the gene are randomly mutated and a can result in many-fold improvements small number of improved variants in activity and substrate specificity are identified by protein expression within a few generations. However, and screening. These selected clones this strategy has the disadvantage of are chopped into fragments of ran- having to examine an extremely high dom length with a restriction enzyme, number of candidates to find the rare express reassembled into full-length se- improvements that are needed as the proteins and quences and amplified using the poly- input for DNA breeding. screen for merase chain reaction, or PCR, creating A more potent variation of the DNA improved a much larger number of new combi- breeding strategy goes by the term function nations. This fragmentation-religation multi-gene shuffling or DNA family process creates novel combinations of breeding; it refers to the recombination the original set of mutations, while of multiple equivalent genes from re- preserving the order of the pieces. The lated species rather than random mu- exponential nature of PCR generates an enormous number of new se- Figure 4. Single-gene breeding starts with the quences in only a few hours. generation of sequence diversity through random mutagenesis. After an initial screen to Generating molecular diversity is far select promising mutants from among thou- best candidates easier than evaluating the performance sands of clones, the best candidates can be of the new molecules. Hundreds of “bred” by cutting and recombining the unique progeny produced by PCR parental sequences to yield “progeny.” A sec- must be analyzed to find the small ond screen identifies new molecules with repeat fraction that can perform the desired even further improvements in function. © 2003 Sigma Xi, The Scientific Research Society. Reproduction with permission only. Contact [email protected]. www.americanscientist.org 2003 November–December 529 same gene from related species Yersinia 196AA 50%

cut and reform using polymerase chain reaction

1402AA 27%

progeny 142AA 37% Citrobacter Enterobacter

screen for improved function

best 181AA candidates 47% repeat Klebsiella

Figure 5. In DNA family breeding, the initial sequence diversity is provided by recombining the same gene from different species (left). Sim- ilar to single-gene breeding, the parental DNA is shuffled into new arrangements while preserving the order of the pieces. An example of this technique is the recombinant beta-lactamase enzyme shown above, which is a mosaic made from portions of the same gene from four distant- ly related microbes. This particular shuffled protein is 270 times more efficient than the best parental sequence. (Courtesy of the authors.) tagenesis of a single gene. This ap- our earlier work with the technique, shuffling are proportional to the size of proach takes advantage of the fact that which we presented in 1998. In that the genome. One example of this tech- while extensive novelty is provided by study, we compared random mutage- nique used Streptomyces bacteria, the cross-species interchange, most of nesis of single genes to multi-gene which are valuable as natural sources the deleterious mutations have long shuffling of an identical set of related of the antibiotic tylosin. Understand- ago been removed by natural selec- sequences. We chose to focus on bacte- ably, drug makers would like to find tion. The reassortment of old, proven rial genes that encode the beta- strains that show higher tylosin ex- mutations yields a higher frequency of lactamase enzyme, which are clinically pression to increase production effi- functional progeny sequences, and be- relevant because these proteins ciency. Using a classic approach based cause multi-gene shuffling starts with inactivate the antibiotic penicillin. The on random point mutation and screen- more than one parental sequence, it ac- starting genes came from four distant- ing, a team of scientists developed cesses a broader range of progenitor ly related microbes and differed in se- Streptomyces variants with a six-fold in- combinations. These attributes make quence by 58 to 69 percent. We found crease in tylosin production. This the process more efficient, minimizing that the best clone generated by ran- achievement required 20 years of mu- loss-of-function mutations so that few- dom mutagenesis showed an eight- tation and selection, and the cumula- er progeny molecules need to be fold increase in beta-lactamase catalyt- tive screening of more than 1,000,000 screened to discover superior per- ic activity. However, when the same mutants. Our lab performed a similar formers. In our lab, we observed that four genes were shuffled, the best search using genome shuffling, and we this variation of DNA breeding yields clone was 33 times better than the ran- found the same improvement—but it striking improvements in complex dom mutation champion—in other was achieved in a single year after properties even with just a few hun- words, a 270-fold increase in the rate of screening only 24,000 mutants. dred progeny. Because screening is the catalysis. This technique is applied more fre- most laborious step in directed evolu- quently to mimic protein evolution in tion, DNA family breeding is a major Widespread Shuffling prokaryotes, which have continuous advance. We can also broaden the technology by genes. We have developed other meth- This kind of directed evolution is applying the concept of DNA breeding ods specifically suited to evolve eu- not only faster—it is also remarkably to entire genomes rather than single karyotic proteins, which are encoded powerful. This can be seen in some of genes; the advantages of such genome by many short stretches of DNA sepa- © 2003 Sigma Xi, The Scientific Research Society. Reproduction with permission only. Contact [email protected]. 530 American Scientist, Volume 91 rated by long noncoding spans. We can genes; these regions make up 75 per- response to the therapeutic protein, be- mimic eukaryotic protein evolution by cent of the human genome. cause the constituent exons will be fa- focusing on those parts of the genome Exons can move around the genome miliar to human immune systems on that actually encode proteins. using a variety of mechanisms. This the prowl for foreign invaders. To understand how the technique form of mutation based on mobile, works, it’s helpful to look at how eu- functional modules is termed exon shuf- Breeding Molecular Medicines karyotic proteins are made. A single fling. It is predisposed to be far more Technological advances are important, protein often resembles a string with useful, in frequency and magnitude, but ultimately they are only tools used many beads, in which each bead per- than random changes in sequence be- to accomplish larger goals. Today, virtu- forms a specific task, such as binding a cause exons or groups of exons fre- ally any human protein, such as a cy- target. These structurally and function- quently encode autonomous functional tokine, growth factor, antibody or en- ally autonomous regions, or domains, domains. zyme, can be cloned, expressed, purified work in combination to execute the Last year, we published a method and rendered administrable as a poten- overall function of the protein. One or for in vitro exon shuffling, once again tial therapeutic. The completion of the more DNA segments called exons, mimicking a natural evolutionary human genome project has enabled which make up only about 1 percent process, by PCR-based recombination scientists to identify hundreds of new of the human genome, encode each do- of multiple exons. This technique proteins with the potential to treat dis- main. Between the exons are large promises to be especially useful in the ease. However, because ideal drug spans of intervening, noncoding in- creation of new therapeutic proteins properties are generally different from trons, which get distinguished from ex- because the starting variation can come those of the native protein, most of ons during RNA splicing. Tracts of so- entirely from human genes, rather than these pharmaceutical candidates will called junk DNA, mostly short from nonhuman species or random require optimization to meet specific repeated sequences that do not encode mutations. We expect this will mini- therapeutic goals. DNA breeding is any protein information, separate the mize the risks of triggering an immune ideally suited for this purpose. It can

654 mutants

nts 26 parents

Figure 6. DNA family breeding of the subtilisin gene illustrates the potential for rapid improvement in protein function. Subtilisin is a protein- degrading enzyme used in laundry detergent and the most highly engineered enzyme in existence. Twenty-six subtilisin genes from various Bacillus microbes were shuffled to yield 654 progeny that were screened for enzyme activity at five commercially relevant conditions. Each clone is shown as a set of concentric circles, with the size of the circle indicating the activity of the enzyme and each assay condition represented by a different color. Several progeny showed simultaneous improvement in multiple properties over the best parental sequences. © 2003 Sigma Xi, The Scientific Research Society. Reproduction with permission only. Contact [email protected]. www.americanscientist.org 2003 November–December 531 bacterial genomes from same species human genes with similar exon structure mediated by the interaction of CD80 with two receptors with very different effects. Binding to one of them increas- es the immune response, which we sought to bolster in order to improve the response to vaccines and to fight cancer and infection. In contrast, when cut and cut and CD80 binds to the other receptor the reform reform immune response is reduced, which would be useful in the treatment of au- toimmune diseases and to create toler- ance for organ transplants. We strove to increase the selectivity of these binding events by shuffling CD80 genes from seven species—human, orang- utan, rhesus monkey, baboon, cat, cow and rabbit—and screening the progeny for preferential binding to one receptor but not the other. The strategy was successful, and the two distinct screen progeny screen progeny types of CD80 variants have behaved for best for best as expected in a variety of in vitro as- candidates candidates says. One of these new molecules is being evaluated further in monkeys. A third area of research in our lab is the development of a vaccine for dengue fever, a viral disease transmit- repeat repeat ted by mosquitoes. Dengue was origi- nally a tropical disease, but it is rapidly spreading throughout the world and Figure 7. Genome shuffling (left) and exon shuffling (right) are two other applications of the has entered the United States. While DNA breeding technique. Genome shuffling is modeled after the natural evolution of prokary- surviving a bout of the disease does otic organisms, whereas exon shuffling mimics eukaryotic protein evolution. In both cases the confer immunity, there are four viral progeny are more likely to have useful properties because the breeding step starts with “proven variants or serotypes, and they each elic- diversity”—natural selection having already removed most deleterious point mutants. it a distinct immune response. Sur- vivors that are reinfected with a differ- enhance desirable biological activities existing drug. After the second round ent serotype are at increased risk of such as binding activity, receptor speci- of shuffling and screening, we isolated developing an often-fatal disease called ficity, circulation half-life, expression clones with a 285,000-fold increase in po- dengue hemorrhagic fever, so dengue level and stability, and reduce undesir- tency compared with the product now vaccines must protect against all four able side effects such as immune-sys- available. DNA sequencing showed serotypes simultaneously. Our ap- tem rejection and toxicity. that the three best progeny each con- proach to this dilemma was to create a One example of the potential for di- sisted of segments from up to five single-protein vaccine: Genes encoding rected evolution to improve existing parental interferons without any amino the antigens from all four variants were drug therapies comes from our work acid point mutations—an important shuffled, and the resulting protein with interferon alpha, variants of advantage because the lack of point progeny was tested for binding to anti- which are encoded by more than 20 mutations decreases the likelihood of bodies that recognize each of the four. genes in humans. Interferons are a an immune response, a common prob- The best vaccine candidates were com- group of cytokine proteins that have a lem with traditional mutants. In stud- plex combinations of the key sites from broad spectrum of anticancer and an- ies using live mice, animals that were all four parental serotypes. These pro- tiviral activities, and human interferon given the novel interferons were fully teins were evaluated in mice for their alpha is already a billion-dollar prod- protected against viral infection, vaccine potential, and several induced uct used to treat these conditions. We whereas mice given the most potent antibody responses that cross-neutral- sought to optimize interferons to com- native interferon enjoyed only partial ized all four dengue serotypes—com- bat specific types of human cancer or protection. pletely preventing viral infection. Can- viruses using DNA breeding. With a Another project focuses on our aim didate vaccines are now progressing to starting pool of 20 interferon alpha to fine tune immune responses by trials in primates. genes, we screened 1,700 shuffled modifying the T-cell surface protein We haven’t yet reached the end of clones with a total of only 68 antiviral CD80. This protein is crucial to the the potential offered by directed evo- assays and found several progeny pro- rapid immune response that targets lution. The application of the technique teins with improved activity on mouse foreign antigens, such as pathogens, to vaccine development is especially cells. The best of these showed specific while ignoring native proteins. The de- promising: By shuffling sequences activity 135,000 times greater than the cision to attack or remain quiescent is from multiple viral subtypes into a sin- © 2003 Sigma Xi, The Scientific Research Society. Reproduction with permission only. Contact [email protected]. 532 American Scientist, Volume 91 Sinclair Stammers/Science Photo Library pseudocolored transmissionelectronmicrographillustratesdenguevirusparticles appears promising:Earlyresultsinmicesuggestthatseveralofthevaccinecandidatespreventinfectionfromallfourserotyp to developaneffective vaccineespeciallysuitedto theapplicationofDNAbreeding.Shuffling theviralantigensintoasingl man beings.Dengueanddenguehemorrhagicfevercanbecausedbyanyoffourdistinctviralvarieties,orserotypes,thism www.americanscientist.org itself, evolving ourmedicalandim- have theability toaffect the treadmill stay inplace.Butforthefirst timewe system mustconstantlyrun inorder to technology andthehuman immune Carroll’s agents. LiketheRedQueenfrom Lewis cannot anticipateallfuture infectious is temporary—theshuffling technique cal samples.Ofcourse,thisadvantage tibiotic-resistant pathogens from clini- natural evolution,asobservedinan- to rapidlypredict thefuture courseof ratory-directed evolution canbeused the pathogenicspectrum. summed diversityofanentire sliceof oculant wouldbeprepared tomeetthe primed withashuffled, multistrainin- drugs. Yet maybeanimmunesystem nization efforts andantimicrobial rieties thatthreaten toevadeimmu- pressure togeneratesubstrainsandva- from, evolutionexertsexceptional us. With aworldofmicrobes toselect ing numberofpathogensthatassault vantage conferred bytheoverwhelm- even helpreverse thetraditionalad- licobacter pylori those causedbytheulcer-inducing and evenbacterialinfectionssuchas pes simplex1and2,footmouth, quer influenza,humanpapilloma,her- gle protein, wemightbeabletocon- Figure 8.Denguefeverisaviraldiseasethatcarriedbythemosquito As wedemonstratedin2001,labo- Alice inWonderland . Thisapproach may , medical © 2003SigmaXi, TheScientificResearch Society. Reproduction He- with permission only. [email protected]. Crameri, A.,S.-A.Raillard, E.Bermudezand Chang, C.-C.,T. T. Chen,B.W. Cox,G.N. Bibliography fertile cornucopia. proteins toflowfrom biotechnology’s opment ofthenextgenerationuseful tion willexponentiallycatalyzedevel- We further thefractionofusefulprogeny. ed-evolution methodologiestoincrease it toowillbeincorporatedintodirect- tein engineeringcontinuestoprogress, and astrue, bottom-up,rationalpro- may beusedinashuffling reaction, tually anycombinationofsequences putable strength ofbothprocesses. Vir- opportunistic, andthisisanindis- on naturalevolution—bothare blindly than thatofitschallengers. mune systemdefensesatafasterrate Kolkman, J.A.,andW. Stemmer. 2001.Direct- Joo, H.,Z.LinandF. H. Arnold.1999.Labora- r genes from diverse speciesacceleratesdi- W. Biotechnology tokine usingmolecularbreeding. P. Dawes, J.Punnonen,W. P. C.Stemmerand DNA breeding isexplicitlymodeled Nature Biotechnology ed evolutionofproteins byexonshuffling. 399:670–673. tochrome P450 hydroxylation. tory evolutionofperoxide-mediated cy- ected evolution.

are optimisticthatdirected evolu- A. Patten.1999.Rapidevolutionofacy-

C. Stemmer. 1998.DNAshuffling of 17:793–797. Nature 19:423–428. Aedes aegypti(left) 391:288–291. (red circles) Nature Nature , aspeciesthatfeedsduringthedayandprefershu- within ahostcell. Zhang, Y.-X., K.Perry, V. A.Vinci, K.Powell, Zhang, J.,G.DawesandW. P. C.Stemmer. Stemmer, W. P. C.1994.Rapidevolutionofa Schmidt-Dannert, C.,D.UmenoandF. H. Orencia, C.,J.S.Yoon, J.E.Ness,W. P. C.Stem- For relevant Web links,consultthisissueof 15:644–646. notypic improvement inbacteria. 2002. Genomeshuffling leadstorapidphe- W. 94:4504–4509. my ofSciencetheUnitedStatesAmerica screening. from agalactosidasebyDNAshuffling and 1997. Evolutionofaneffective fucosidase 370:389–391. protein invitro byDNAshuffling. Biotechnology carotenoid biosyntheticpathways. Arnold. 2000.Molecularbreeding of Structural Biology ed evolutionandstructural analysis. emergence ofantibioticresistance bydirect- mer andR.C.Stevens.2001.Predicting the http://www

C. StemmerandS.B.DelCardayré. 2003 November–December 533 mrcnSinitOnline: American Scientist IssueT Proceedings oftheNationalAcade- 18:750–753. .americanscientist.or OC/issue/406 8:238–242. e proteininoculant akes theeffort es. Atright,a g/ Nature Nature Nature Nature

London School of Hygiene and Tropical Medicine/Science Photo Library