We have demonstrated that saturation mutagenesis libraries with no codon redundancy and amino acid biases could be easily constructed by our newly introduced small-intelligent strategy. Since the only difference between small-intelligent primer and the conventional NNS primer is the codon composition, it is reasonable to assume that any PCR strategies that work with NNS primer are adoptable with small-intelligent primer. Thus, the small-intelligent strategy is feasible for the construction of high-quality saturation libraries and could serve as a good substitute for NNS randomization.
In comparison with the MAX system, the small-intelligent system offers an alternative for producing libraries with the same quality as MAX does, but are less complex and less expensive in most cases, since there are no requirements for ligation, restriction digestion, as well as template synthesis, and fewer oligonucleotides are needed in the small-intelligent system relative to the MAX system, except for the case where multiple contiguous sites are randomized. Most importantly, our system would allow for randomization of multiple contiguous amino acids, although more oligonucleotides would need to be synthesized. However, this situation could be improved by applying the iterative CASTing method (23).
In addition to full randomization, the small-intelligent system should also allow us to perform any restricted randomizations using a specific primer set, and such primer sets could be easily achieved with the assistance of DC-Analyzer. Taken together, the small-intelligent strategy could serve as a simple and valuable tool to construct high-quality focused mutagenesis libraries.
This research was supported by the National Natural Science Foundation of China (no. 20872014).
The authors declare no competing interests.
Address correspondence to Lixia Tang, School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu 610054, China. e-mail: [email protected]
1.) Arnold, F.H., and A.A. Volkov. 1999. Directed evolution of biocatalysts. Curr. Opin. Chem. Biol. 3:54-59. 2.) Turner, N.J. 2009. Directed evolution drives the next generation of biocatalysts. Nat. Chem. Biol. 5:567-573. 3.) Stemmer, W.P. 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389-391. 4.) Crameri, A., S.A. Raillard, E. Bermudez, and W.P. Stemmer. 1998.. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391:288-291. 5.) Lutz, S. 2010. Beyond directed evolution—semi-rational protein engineering and design. Curr. Opin. Biotechnol. 21:734-743. 6.) Chica, R.A., N. Doucet, and J.N. Pelletier. 2005. Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. Curr. Opin. Biotechnol. 16:378-384. 7.) Richard, J.F., and G.W. Huisman. 2008. Enzyme optimization: moving from blind evolution to statistical exploration of sequence-function space. Trends Biotechnol. 26:132-138. 8.) Reetz, M.T., L.W. Wang, and M. Bocola. 2006. Directed evolution of enantioselective enzymes: iterative cycles of casting for probing protein-sequence space. Angew. Chem. Int. Ed. 118:1258-1263. 9.) Banáš, P., M. Otyepka, P. Jeřábek, M. Petřek, and J. Damborský. 2006. Mechanism of enhanced conversion of 1,2,3-trichloropropane by mutant haloalkane dehalogenase revealed by molecular modeling. J. Comput. Aided Mol. Des. 20:375-383. 10.) Korkegian, A., M.E. Black, D. Baker, and B.L. Stoddard. 2005. Computational thermo-stabilization of an enzyme. Science 308:857-860. 11.) Reetz, M.T., J.D. Carballeira, and A. Vogel. 2006. Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew. Chem. Int. Ed. 45:7745-7751. 12.) Pupko, T., R.E. Bell, I. Mayrose, F. Glaser, and N. Ben-Tal. 2002. Rate4Site: an algorithmic tool for the identification of functional regions in proteins by surface mapping of evolutionary determinants within their homologues. Bioinformatics 18:S71-S77. 13.) Nimrod, G., F. Glaser, D. Steinberg, N. Ben-Tal, and T. Pupko. 2005. In silico identification of functional regions in proteins. Bioinformatics 21:1328-1337. 14.) Neylon, C. 2004. Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res. 32:1448-1459. 15.) Neuner, P., R. Cortese, and P. Monaci. 1998. Codon-based mutagenesis using dimer-phosphoramidites. Nucleic Acids Res. 26:1223-1227. 16.) Virnekäs, B., L.M. Ge, A. Pluckthun, K.C. Schneider, G. Wellnhofer, and S.E. Moroney. 1994. Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 22:5600-5607. 17.) Hughes, M.D., D.A. Nagel, A.F. Santos, A.J. Sutherland, and A.V. Hine. 2003. Removing the redundancy from randomised gene libraries. J. Mol. Biol. 331:973-979. 18.) Reetz, M.T., S. Prasad, J.D. Carballeira, Y. Gumulya, and M. Bocola. 2010. Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods. J. Am. Chem. Soc. 132:9144-9152. 19.) Zheng, L., U. Baumann, and J.-L. Reymond. 2004. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32:e115. 20.) Tseng, W., J. Lin, T. Wei, and T. Fang. 2008. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal. Biochem. 375:376-378. 21.) Sanchis, J., L. Fernández, J.D. Carballeira, J. Drone, Y. Gumulya, H. Höbenreich, D. Kahakeaw, S. Kille. 2008. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Appl. Microbiol. Biotechnol. 81:387-397. 22.) Wang, J., S. Zhang, H. Tan, and Z. Zhao. 2007. PCR-based strategy for construction of multi-site-saturation mutagenic expression library. J. Microbiol. Methods 71:225-230. 23.) Reetz, M.T., M. Bocola, J.D. Carballeira, D. Zha, and A. Vogel. 2005. Expanding the range of substrates acceptance of enzymes: combinatorial active-site saturation test. Angew. Chem. 117:4264-4268. 24.) Mena, M.A., and P.S. Daugherty. 2005. Automated design of degenerate codon libraries. Protein Eng. Des. Sel. 18:559-561. 25.) Firth, A.E., and W.M. Patrick. 2008. GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries. Nucleic Acids Res. 36:W281-W285.