Abstract
Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (stability, activity, immunogenicity, etc.). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries—a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 107 variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and novelty as well as library construction technique, and identify optimal libraries for experimental evaluation.
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References
Cadwell, R.C., Joyce, G.F.: Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992)
Chen, C.Y., Georgiev, I., Anderson, A.C., Donald, B.R.: Computational structure-based redesign of enzyme activity. PNAS 106, 3764–3769 (2009)
Fox, R., et al.: Improving catalytic function by ProSAR-driven enzyme evolution. Nat. Biotechnol. 25, 338–344 (2007)
Fukuda, H., et al.: Reconstitution of the isobitene-forming reaction catalyzed by cytochrome p450 and p450 reductase from Rhodotorula minuta: Decarboxylation with the formation of isobutene. Biochem. Bioph. Res. Co. 201, 516–522 (1994)
Griswold, K.E., Aiyappan, N.S., Iverson, B.L., Georgiou, G.: The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase. J. Mol. Biol. 364, 400–410 (2006)
Harding, F.A., et al.: A beta-lactamase with reduced immunogenicity for the targeted delivery of chemotherapeutics using antibody-directed enzyme prodrug therapy. Mol. Cancer. Ther. 4, 1791–1800 (2005)
He, L., Friedman, A.M., Bailey-Kellogg, C.: Pareto optimal protein design. In: 3dsig: Structural Bioinformatics and Computational Biophysics, pp. 69–70 (2010)
Herman, A., Tawfik, D.S.: Incorporating synthetic oligonucleotides via gene reassembly (ISOR): a versatile tool for generating targeted libraries. Protein Eng. Des. Sel. 20, 219–226 (2007)
Hiraga, K., Arnold, F.: General method for sequence-independent site-directed chimeragenesis. J. Mol. Biol. 330, 287–296 (2003)
Jackel, C., Bloom, J.D., Kast, P., Arnold, F.H., Hilvert, D.: Consensus protein design without phylogenetic bias. J. Mol. Biol. 399, 541–546 (2010)
Jiang, L., et al.: De novo computational design of retro-aldol enzymes. Science 319(5868), 1387–1391 (2008)
la Grange, D.C., den Haan, R., van Zyl, W.H.: Engineering cellulolytic ability into bioprocessing organisms. Appl. Microbiol. Biotechnol. 87, 1195–1208 (2010)
Levin, A.M., Murase, K., Jackson, P.J., Flinspach, M.L., Poulos, T.L., Weiss, G.A.: Double barrel shotgun scanning of the Caveolin-1 scaffolding domain. ACS Chem. Biol. 2, 493–500 (2007)
Marco, A.M., Daugherty, P.S.: Automated design of degenerate codon libraries. Protein Eng. Des. Sel. 18, 559–561 (2005)
Meyer, M., Hochrein, L., Arnold, F.: Structure-guided SCHEMA recombination of distantly related beta-lactamases. Protein Eng. Des. Sel. 19, 563–570 (2006)
Nelson, A., Reichert, J.M.: Development trends for therapeutic antibody fragments. Nat. Biotech. 27, 331–337 (2009)
Otey, C., Landwehr, M., Endelman, J., Hiraga, K., Bloom, J., Arnold, F.: Structure-guided recombination creates an artificial family of cytochromes P450. PLoS Biol. 4, e112 (2006)
Pantazes, R., Saraf, M., Maranas, C.: Optimal protein library design using recombination or point mutations based on sequence-based scoring functions. Protein Eng. Des. Sel. 20, 361–373 (2007)
Parker, A.S., Griswold, K., Bailey-Kellogg, C.: Optimization of therapeutic proteins to delete T-cell epitopes while maintaining beneficial residue interactions. In: Proc. CSB, pp. 100–113 (2010)
Parker, A.S., Zheng, W., Griswold, K., Bailey-Kellogg, C.: Optimization algorithms for functional deimmunization of therapeutic proteins. BMC Bioinf. 11, 180 (2010)
Pierce, N., Winfree, E.: Protein design is NP-hard. Protein Eng. 15, 779–782 (2002)
Reetz, M.T., Carballira, J.: Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protocols 2, 891–903 (2007)
Reetz, M.T., Kahakeaw, D., Lohmer, R.: Addressing the numbers problem in directed evolution. ChemBioChem. 9, 1797–1804 (2008)
Russ, W.P., Lowery, D.M., Mishra, P., Yaffee, M.B., Ranganathan, R.: Natural-like function in artificial WW domains. Nature 437, 579–583 (2005)
Saraf, M.C., Gupta, A., Maranas, C.D.: Design of combinatorial protein libraries of optimal size. Proteins 60, 769–777 (2005)
Saraf, M.C., Horswill, A.R., Benkovic, S.J., Maranas, C.D.: FamClash: A method for ranking the activity of engineered enzymes. PNAS 12, 4142–4147 (2004)
Socolich, M., Lockless, S.W., Russ, W.P., Lee, H., Gardner, K.H., Ranganathan, R.: Evolutionary information for specifying a protein fold. Nature 437, 512–518 (2005)
Stemmer, W.P.C.: DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. PNAS 91, 10747–10751 (1994)
Treynor, T., Vizcarra, C., Nedelcu, D., Mayo, S.: Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function. PNAS 104, 48–53 (2007)
Voigt, C.A., Martinez, C., Wang, Z.G., Mayo, S.L., Arnold, F.H.: Protein building blocks preserved by recombination. Nat. Struct. Biol. 9, 553–558 (2002)
Ye, X., Friedman, A.M., Bailey-Kellogg, C.: Hypergraph model of multi-residue interactions in proteins: Sequentially–constrained partitioning algorithms for optimization of site-directed protein recombination. J. Comput. Biol. 14, 777–790 (2007); In: Apostolico, A., Guerra, C., Istrail, S., Pevzner, P.A., Waterman, M. (eds.) RECOMB 2006. LNCS (LNBI), vol. 3909, pp. 15–29. Springer, Heidelberg (2006)
Zhang, J., Campbell, R., Ting, A., Tsien, R.: Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell. Biol. 3, 906–918 (2002)
Zheng, W., Friedman, A., Bailey-Kellogg, C.: Algorithms for joint optimization of stability and diversity in planning combinatorial libraries of chimeric proteins. J. Comput. Biol. 16, 1151–1168 (2009); In: Vingron, M., Wong, L. (eds.) RECOMB 2008. LNCS (LNBI), vol. 4955, pp. 300–314. Springer, Heidelberg (2008)
Zheng, W., Griswold, K., Bailey-Kellogg, C.: Protein fragment swapping: A method for asymmetric, selective site-directed recombination. J. Comput. Biol. 17, 459–475 (2010); In: Batzoglou, S. (ed.) RECOMB 2009. LNCS, vol. 5541, pp. 321–338. Springer, Heidelberg (2009)
Zheng, W., Ye, X., Friedman, A., Bailey-Kellogg, C.: Algorithms for selecting breakpoint locations to optimize diversity in protein engineering by site-directed protein recombination. In: Proc. CSB, pp. 31–40 (2007)
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Parker, A.S., Griswold, K.E., Bailey-Kellogg, C. (2011). Optimization of Combinatorial Mutagenesis. In: Bafna, V., Sahinalp, S.C. (eds) Research in Computational Molecular Biology. RECOMB 2011. Lecture Notes in Computer Science(), vol 6577. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-20036-6_29
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DOI: https://doi.org/10.1007/978-3-642-20036-6_29
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