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Directed evolution library creation: Methods and protocols. Methods in molecular biology, Vol. 231, edited by Frances H. Arnold and George Georgiou. 2003. Totowa, NJ: Humana Press. 224 pages.
Directed molecular evolution of proteins: Or how to improve enzymes for biocatalysis, edited by Susanne Brackmann and Kay Johnsson. 2002. Weinheim, Germany: Wiley-VCH. 357 pages.
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76 100, Israel
Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04754204.
In the last three years, the combinatorial approach toward protein engineering has gathered momentum and success, thus defining a new discipline of research that is generally referred to as directed evolution of proteins. Like any other new scientific discipline, the early stages of development were accompanied with doubts as to its scope and general applicability. (Except that, in nature, this strategy has been operating for about 4 billion years with some truly stunning results.) During this "wait and see" era, directed evolution was pursued by a relatively small number of groups that specialized in this area and in the development of new methodologies such as phage display or DNA shuffling. These methodologies are now being applied by many groups, for which directed evolution is merely a powerful tool and not the essence of research. The evolution of this discipline is apparent in the constantly growing number of journal articles (Fig. 1
), as well as in the publication of the three new books reviewed here.
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-galactosidase variants evolved in Escherichia coli). Directed evolution in the laboratory has seen a burst with the development of nucleic acids in vitro evolution (SELEX), which has since led to numerous aptamers, ribozymes, and DNA enzymes. The area of directed evolution of proteins has evolved more gradually, the main reason being that the simple principle behind nucleic acid evolution—namely, that the same molecule comprises the gene and the enzyme, or the gene and the binding entity (aptamers)—does not apply to proteins. Here, linking genotype to phenotype requires a living cell or rather complex in vitro strategies. Directed evolution of proteins also requires a much more diverse array of methodologies for creating genetic diversity and for selecting this diversity for the desired function. These methodologies are the subject of the first two volumes described here. The volumes are part of the well-established series Methods in Molecular Biology by Humana Press (edited by John M. Walker), and cover library creation methods and screening and selection methods, respectively. The editors of these two books followed the useful format of the series that provides many detailed protocols (including notes that address the tricky parts of the protocol) accompanied by short introductions and a few chapters that follow a review, rather than protocol, style.
There seems to be, however, a significant difference in the generality, scope, and usefulness of the two volumes—a difference that is best described by the cynical version of the "central dogma of molecular biology"—DNA gives RNA gives Troubles. The making of gene libraries is a clearly defined area where the same protocol is applicable to almost every gene. In contrast, screening and selection methods (as well as expression of the proteins to be screened) need to be specifically tailored for each protein and function. Hence the editors’ inevitable apology in the preface of the second volume toward those readers who will not find a screen for their favorite enzyme. Indeed, the first volume provides a comprehensive coverage of the different approaches for creating genetic diversity. Most of the useful methodologies are in vitro and rely primarily on PCR (polymerase chain reaction). Nevertheless, in vivo methods (e.g., use of mutator bacterial strains) are very well covered. The volume also contains a very useful section on the "analysis of library diversity." In my view, the only generally useful method that is regrettably not covered is the use of wobble-base PCR for random mutagenesis. The extensive coverage and general applicability and clarity of the protocols make the first volume a must for all groups interested in the area, as well as institutional libraries. The second volume covers a broad range of enzymes, screens, and selections. Regarding the latter, there is a clear and deliberate focus on in vivo methods, including genetic selections. This volume offers no coverage of purely in vitro methods (that may justify an independent volume) and only partial and sporadic coverage of semi in vitro methods such as phage display. In summary, the second volume can certainly be of much utility, primarily for those entering the field and wishing to survey various approaches and methodologies, albeit there will be only a few cases in which the detailed protocols offered in it can be applied "as is."
The third book, Directed Molecular Evolution of Proteins, does not focus on methodology. Rather, it provides 14 chapters that describe various aspects of the field in a review style. Although there is a certain degree of overlap in coverage between this book and the previous two, they are by and large complementary. It has to be said that, as is the case with most edited books, there is a considerable variability in the scope, aims, and coverage of the different chapters. Only a few chapters provide a broad overview that does not focus on the authors’ own work. These comprise, in my view and in the view of several others who have seen this book, the most useful part of the book. Such a perspective as provided by Lutz and Benkovic (engineering protein evolution) on the strategies applied by nature to evolve new proteins is indispensable, as it has no parallels in one article or textbook. Similar in style is the chapter by Schuster on the theoretical and computational aspects of molecular evolution and the last chapter on generation of enzymes with new substrate specificities by Bornscheuer. These chapters (and the "engineering protein evolution" chapter in particular) can serve as textbook material and for advanced course reading. The remaining chapters focus on the work of a single laboratory or even a single enzyme. These are useful primarily for those interested in a particular system, and their content largely overlaps published journal articles. The emphasis is on enzymes and screen or selections that make use of living cells (in vivo). Coverage of these specific topics is inevitably partial and at times sporadic, with considerable repetition between chapters (e.g., in describing library making). Some "classical" areas are covered (e.g., evolution towards stability in organic solvents) whereas others (e.g., evolution towards thermostability) are not. Despite these drawbacks, this book is recommended, even if only for the few broad, textbook-style chapters that cover for the absence of up-to-date books in the area of protein evolution.
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