last updated 12/2003
The application of molecular evolution to proteins has proven to be an effective method for engineering proteins with improved properties. Molecular evolution works through cycles of (a) creating a library of gene variations and (b) identifying by selection or screening those rare members of the library which code for proteins that have an improvement in function. Depending on the method of library construction and the methods available for selection and screening, libraries of up to 109 or more variants can be constructed and evaluated. However, even this seemingly large number of variants is but a minutia of the possible number of the 20300 possible sequences that could code for an average size protein of 300 amino acids. Thus, since one can create only an infinitesimal fraction of the possible variations of a gene or genes, the more one can tailor the library to be as rich in function as possible, the higher the probability of creating and identifying a protein with improved properties.
Common methods of library construction, including error-prone PCR and DNA shuffling, create diversity by changing the amino acid sequences. In contrast, incremental truncation, a method for creating a library of every one base truncation of dsDNA, creates diversity by changing the length of a gene. Incremental truncation libraries can be created by time dependent Exo III digestions (1) or by the incorporation of a-phosphorothioate dNTPs (4). The combination of two incremental truncation libraries, called ITCHY libraries (3), creates diversity by fusing two gene fragments. Performing ITCHY on a single gene generates libraries of proteins with internal deletions and duplications whereas performing ITCHY between two different genes generate libraries of fusion proteins in a DNA-homology independent fashion. Both strategies, as well as an incremental truncation-like method called SHIPREC (Sieber et al., 2001), have the potential to create proteins with improved or novel properties as well as to generate artificial families for in vitro recombination in a method called SCRATCHY (6). However, different methods for creating incremental truncation libraries produce libraries with different distributions of truncation lengths (7). Incremental truncation and ITCHY have a number of demonstrated and potential applications apart from directed evolution including defining minimal functional units, identifying independent folding units and assigning function to domains and subdomains.
(9) Kawarasaki, Y., K.E. Griswold, J.D. Stevenson , T. Selzer, S.J. Benkovic, B.L. Iverson, and G. Georgiou. "Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers." Nucleic Acids Research 31(21), e126 (2003). PDF
(8) Lee, S.G., S. Lutz S, and S.J. Benkovic. "On the structural and functional modularity of glycinamide ribonucleotide formyltransferases." Protein Science 12(10), 2206-2214 (2003). PDF(7) Ostermeier, M. "Theoretical distribution of truncation lengths in incremental truncation libraries." Biotechnology and Bioengineering 82, 564-577 (2003). PDF
(6) Lutz, S., M. Ostermeier, G. Moore, C. Maranas, and S.J. Benkovic. "Creating multiple-crossover DNA libraries independent of sequence identity." Proceedings of the National Academy of Science USA 98, 11248 (2001). PDF supplemental information
(5) Ostermeier, M. and S.J. Benkovic. "A method for the construction of hybrid gene libraries involving the circular permutation of DNA." Biotechnology Letters 23, 303 (2001). PDF
(4) Lutz, S., M. Ostermeier, and S.J. Benkovic. "Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides." Nucleic Acids Research 29, e16 (2001). PDF
(3) Ostermeier, M., J.H. Shim, and S.J. Benkovic. "A combinatorial approach to hybrid enzymes independent of DNA homology." Nature Biotechnology 17, 1205 (1999). PDF news & views
(2) Ostermeier, M.,
A.E. Nixon, and S.J. Benkovic. "Incremental truncation as a strategy in the engineering of novel catalysts." Biorganic and Medicinal Chemistry 7, 2139 (1999).
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(1) Ostermeier, M.,
A.E. Nixon, J.H. Shim, and S.J. Benkovic. "Combinatorial protein engineering
by incremental truncation." Proceedings of the National Academy of
Science USA 96, 3562 (1999).
PDF
Paschon, D.E. and M. Ostermeier. "Construction of protein fragment complementation libraries using incremental truncation." Methods Enzymol. in press (2004).
Lutz, S. and M. Ostermeier. "Preparation of SCRATCHY hybrid protein libraries: size- and in-frame selection of nucleic acid sequences" Methods in Molecular Biology 231, 143 (2003).
Ostermeier, M., S. Lutz. and S. J. Benkovic "Generation of protein fragment libraries by incremental truncation." In: Golemis, E.A. (ed) Protein-Protein Interactions: A Molecular Cloning Manual. Cold Spring Harbor Laboratory Press (Plainview, NY, 2002).
Note: there were a couple of minor but potentially confusing errors that were made in the editing of the protocols of this chapter. The corrections are listed here.