The Immune System as a Tool for Directed Evolution

PULLING OUT A PLUM:© 2004 National Academy of ScienceStereoview of mutation loci in mPlum based on the crystal structure of DsRed. Red: the RFP chromophore; yellow: emission shift mutations; gray: neutral mutations.A team of researchers at the University of California, San Diego, has harnessed the body's ability to tweak antibody genes to drive protein evolution in the lab. The group, led by Howard Hughes Medical Institute Investigator Roger Tsien, used the technique to induce the development of novel fluorescent properties in a red-fluorescent protein.1The new method, which is a departure from other protein-manipulation strategies used in the laboratory, derives its power from somatic hypermutation (SHM). B cells use SHM to diversify antibody genes by introducing point mutations in immunoglobulin-variable regions.Protein manipulation is traditionally accomplished using approaches such as site-directed mutagenesis and rational design. These techniques are time-consuming and unreliable, however, because functional changes often stem from multiple mutations that are difficult to predict. Newer strategies, collectively called "directed evolution," are more flexible but can also prove complicated.Directed evolution involves generating a library of mutants via random mutagenesis, DNA shuffling, and/or recombination of a gene of interest, and screening the resulting mutants for a desired property. Clones that make the first cut are subjected to subsequent rounds of mutagenesis until a candidate displaying the required attributes is identified.2"You have to use far more organisms even than you have DNA, because you want no organism to pick up more than one mutant copy. Then you have to grow up the organisms, find out which of them [expresses] the gene you care about, find the few bacteria or yeast, or whatever systems have the best properties out of the millions or so variants that you've generated," says Tsien. The process can take up to a month even for the most skilled technician.In vivo mutagenesis using ultraviolet radiation or harsh chemicals would avoid these problems, but can attack the whole genome rather than just the gene of interest, thereby endangering or destroying the cell. Tsien's new SHM-based technique, in contrast, "actually takes advantage of the power of the immune system of our body to create diversity in proteins," says first author Lei Wang.The researchers ligated the gene for a monomeric red-fluorescent protein (mRFP1.2) into a retroviral vector containing a tetracycline-activated promoter. They expressed the gene in the human B-cell line, Burkitt lymphoma Ramos, using the tetracycline derivative doxycycline. They then screened clones for improvements in photostability and far-red emissions via fluorescence-activated cell sorting. All the mutants displayed higher photostability than the parent protein; after 23 iterations, a mutant dubbed mPlum with an emission wavelength 37 nm longer than mRFP1.2 was isolated.Wang emphasizes the ease of the approach to create a diverse library. "In our case the mammalian cells themselves generate such diversity, you don't need to use anything else [other] than antibiotic to induce the expression of your protein, and then start the mutation process," he says.Frances Arnold, a California Institute of Technology researcher who uses directed evolution to design novel enzymes, calls the technique a "genuine advance" and adds that she would definitely consider using it if her lab worked with proteins in mammalian cells. "Techniques are a dime a dozen, but good techniques like this one are not," she says.Susanne Brakmann, a biologist at the University of Leipzig who also uses directed evolution, agrees that Tsien and Wang's research shows SHM is efficient at mutating a target gene. She adds that some researchers may be reluctant to use the method initially because the precise muta-genesis mechanism is not fully understood.Tsien says much remains to be learned about how mutations occur via SHM in nonantibody genes. "It turns out that the most successful colony was one in which the gene happened by chance to have fallen right next to antibody genes. And so there may be other signals due to antibody genes that make the process a little bit more effective."

The Immune System as a Tool for Directed Evolution

A team of researchers at the University of California, San Diego, has harnessed the body's ability to tweak antibody genes to drive protein evolution in the lab.

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