Survey of Protein Engineering Strategies
互联网
1680
- Abstract
- Table of Contents
- Figures
- Literature Cited
Abstract
Protein engineering is altering the structure of a protein to improve or change its properties. This unit summarizes concepts for protein engineering using rational design, directed evolution, and combinations of them. Different strategies are presented for identifying the best mutagenesis method, how to identify desired variants by screening or selection, and examples for successful applications are given. This should enable researchers to choose the most promising tools to solve their protein engineering challenges. Curr. Protoc. Protein Sci. 66:26.7.1?26.7.14. © 2011 by John Wiley & Sons, Inc.
Keywords: protein engineering; rational protein design; directed evolution; biocatalysis
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Current Strategies to Engineer Proteins
- Strategies to Engineer Specific Properties
- Mutagenesis Methods
- Conclusions
- Acknowledgements
- Literature Cited
- Figures
- Tables
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryMaterials
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryFigures
-
Figure 26.7.1 The native, folded conformation of protein (N) equilibrates with the denatured, unfolded conformation (D). The free energy of unfolding, ΔGD‐N , typically favors the folded conformation by 5‐15 kcal/mol, but this value changes under different conditions. The denatured conformation can irreversibly form insoluble aggregates. Abbreviations: aa, amino acid; E, enantioselectivity. View Image -
Figure 26.7.2 (A ) An engineered nitrilase catalyzes a key enantioselective step in synthesis of an atorvastatin (Lipitor) precursor under industrial scale conditions. (B ) Screening for nitrilase variants with increased enantioselectivity used mass spectrometry and 15 N‐labeled pseudo‐prostereogenic 3‐hydroxyglutaronitrile. View Image -
Figure 26.7.3 An engineered ( R )‐transaminase catalyzes the highly efficient enantioselective formation of a chiral amine for the synthesis of Sitagliptin. The process is superior to the chemical asymmetric reduction. View Image
Videos
Literature Cited
| Arnold, F.H. and Georgiou, G., eds. 2003. Directed Enzyme Evolution: Screening and Selection Methods. Methods in Molecular Biology. Humana Press, Totowa, N.J. | |
| Bartsch, S., Kourist, R. and Bornscheuer, U.T. 2008. Complete inversion of enantioselectivity towards acetylated tertiary alcohols by a double mutant of a Bacillus subtilis esterase. Angew. Chem. Int. Ed. 47:1508‐1511. | |
| Bergeron, S., Chaplin, D.A., Edwards, J.H., Ellis, B.S.W., Hill, C.L., Holt‐Tiffin, K., Knight, J.R., Mahoney, T., Osborne, A.P., and Ruecroft, G. 2006. Nitrilase‐catalysed desymmetrisation of 3‐hydroxyglutaronitrile: Preparation of a statin side‐chain intermediate. Org. Proc. Res. Dev. 10:661‐665. | |
| Bloom, J.D., Labthavikul, S.T., Otey, C.R., and Arnold, F.H. 2006. Protein stability promotes evolvability. Proc. Natl. Acad. Sci. U.S.A. 103:5869‐5874. | |
| Bloom, J.D., Romero, P.A., Lu, Z., and Arnold, F.H. 2007. Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution. Biol. Direct. 2:17. | |
| Bocola, M., Schulz, F., Leca, F., Vogel, A., Fraaije, M.W., and Reetz, M.T. 2005. Converting phenylacetone monooxygenase into phenylcyclohexanone monooxygenase by rational design: Towards practical Baeyer‐Villiger monooxygenases. Adv. Synth. Catal. 347:979‐986. | |
| Bornscheuer, U.T. and Kazlauskas, R.J. 2004. Catalytic promiscuity in biocatalysis: Using old enzymes to form new bonds and follow new pathways. Angew. Chem. Int. Ed. 43:6032‐6040. | |
| Bornscheuer, U.T., Altenbuchner, J., and Meyer, H.H. 1998. Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of Epothilones. Biotechnol. Bioeng. 58:554‐559. | |
| Brakmann, S. and Schwienhorst, A., eds. 2004. Evolutionary Methods in Biotechnology: Clever Tricks for Directed Evolution. Wiley‐VCH, Weinheim, Germany. | |
| Cadwell, R.C. and Joyce, G.F. 1992. Randomization of genes by PCR mutagenesis. PCR Meth. Appl. 2:28‐33. | |
| Carr, R., Alexeeva, M., Dawson, M.J., Gotor‐Fernandez, V., Humphrey, C.E., and Turner, N.J. 2005. Directed evolution of an amine oxidase for the preparative deracemisation of cyclic secondary amines. ChemBioChem 6:637‐639. | |
| Chen‐Goodspeed, M., Sogorb, M.A., Wu, F., and Raushel, F.M. 2001. Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues. Biochemistry 40:1332‐1339. | |
| Cherry, J.R., Lamsa, M.H., Schneider, P., Vind, J., Svendsen, A., Jones, A., and Pedersen, A.H. 1999. Directed evolution of a fungal peroxidase. Nat. Biotechnol. 17:379‐384. | |
| Desai, A.A. 2011. Sitagliptin manufacture: A compelling tale of green chemistry, process intensification, and industrial asymmetric catalysis. Angew. Chem. Int. Ed. 50:1974‐1976. | |
| DeSantis, G., Wong, K., Farwell, B., Chatman, K., Zhu, Z., Tomlinson, G., Huang, H., Tan, X., Bibbs, L., Chen, P., Kretz, K., and Burk, M.J. 2003. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J. Am. Chem. Soc. 125:11476‐11477. | |
| DeSantis, G., Zhu, Z., Greenberg, W.A., and Burk, M.J. 2002. An enzyme library approach to biocatalysis: Development of nitrilases for enantioselective production of carboxylic acid derivatives. J. Am. Chem. Soc. 124:9024‐9025. | |
| Ditursi, M.K., Kwon, S.J., Reeder, P.J., and Dordick, J.S. 2006. Bioinformatics‐driven, rational engineering of protein thermostability. Prot. Eng. Des. Sel. 19:517‐524. | |
| Estell, D.A., Graycar, T.P., and Wells, J.A. 1985. Engineering an enzyme by site‐directed mutagenesis to be resistant to chemical oxidation. J. Biol. Chem. 260:6518‐6521. | |
| Fox, R.J., Davis, S.C., Mundorff, E.C., Newman, L.M., Gavrilovic, V., Ma, S.K., Chung, L.M., Ching, C., Tam, S., Muley, S., Grate, J., Gruber, J., Whitman, J.C., Sheldon, R.A., and Huisman, G.W. 2007. Improving catalytic function by ProSAR‐driven enzyme evolution. Nat. Biotechnol. 25:338‐344. | |
| Gray, K.A., Zhao, L., and Emptage, M. 2006. Bioethanol. Curr. Opin. Chem. Biol. 10:141‐146. | |
| Guerois, R., Nielsen, J.E., and Serrano, L. 2002. Predicting changes in the stability of proteins and protein complexes: A study of more than 1000 mutations. J. Mol. Biol. 320:369‐387. | |
| Gupta, R.D. and Tawfik, D.S. 2008. Directed enzyme evolution via small and effective neutral drift libraries. Nat. Methods 5:939‐942. | |
| Horsman, G.P., Liu, A.M., Henke, E., Bornscheuer, U.T., and Kazlauskas, R.J. 2003. Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3‐bromo‐2‐methylpropanoate and ethyl 3‐phenylbutyrate. Chem. Eur. J. 9:1933‐1939. | |
| Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Rothlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F. 3rd, Hilvert, D., Houk, K.N., Stoddard, B.L., and Baker, D. 2008. De novo computational design of retro‐aldol enzymes. Science 319:1387‐1391. | |
| Jochens, H. and Bornscheuer, U.T. 2010. Natural diversity to guide focused directed evolution. ChemBioChem 11:1861‐1866. | |
| Jochens, H., Stiba, K., Savile, C., Fujii, R., Yu, J.G., Gerassenkov, T., Kazlauskas, R.J., and Bornscheuer, U.T. 2009. Converting an esterase into an epoxide hydrolase. Angew. Chem. Int. Ed. 48:3532‐3535. | |
| Jochens, H., Aerts, D., and Bornscheuer, U.T. 2010. Thermostabilization of an esterase by alignment‐guided focused directed evolution. Prot. Eng. Des. Sel. 23:903‐909. | |
| Johannes, T.W., Woodyer, R.D., and Zhao, H. 2005. Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration. Appl. Environ. Microbiol. 71:5728‐5734. | |
| Kazlauskas, R.J. 2005. Enhancing catalytic promiscuity for biocatalysis. Curr. Opin. Chem. Biol. 9:195‐201. | |
| Kazlauskas, R.J. and Bornscheuer, U.T. 2009. Finding better protein engineering strategies. Nat. Chem. Biol. 5:526‐529. | |
| Kurtzman, A.L., Govindarajan, S., Vahle, K., Jones, J.T., Heinrichs, V., and Patten, P.A. 2001. Advances in directed protein evolution by recursive genetic recombination: Applications to therapeutic proteins. Curr. Opin. Biotechnol. 12:361‐370. | |
| Lehmann, M., Kostrewa, D., Wyss, M., Brugger, R., D'Arcy, A., Pasamontes, L., and van Loon, A.P. 2000a. From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Prot. Eng. 13:49‐57. | |
| Lehmann, M., Pasamontes, L., Lassen, S.F., and Wyss, M. 2000b. The consensus concept for thermostability engineering of proteins. Biochim. Biophys. Acta 1543:408‐415. | |
| Lehmann, M., Loch, C., Middendorf, A., Studer, D., Lassen, S.F., Pasamontes, L., van Loon, A.P., and Wyss, M. 2002. The consensus concept for thermostability engineering of proteins: Further proof of concept. Prot. Eng. 15:403‐411. | |
| Leung, D.W., Chen, E., and Goeddel, D.V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11‐15. | |
| Lewis, J.C., Mantovani, S.M., Fu, Y., Snow, C.D., Komor, R.S., Wong, C.H., and Arnold, F.H. 2010. Combinatorial alanine substitution enables rapid optimization of cytochrome P450BM3 for selective hydroxylation of large substrates. ChemBioChem 11:2502‐2505. | |
| Liao, J., Warmuth, M.K., Govindarajan, S., Ness, J.E., Wang, R.P., Gustafsson, C., and Minshull, J. 2007. Engineering proteinase K using machine learning and synthetic genes. BMC Biotechnology 7:16. | |
| Lutz, S. and Patrick, W.M. 2004. Novel methods for directed evolution of enzymes: Quality, not quantity. Curr. Opin. Biotechnol. 15:291‐297. | |
| Lutz, S. and Bornscheuer, U.T., eds. 2009. Protein Engineering Handbook. Wiley‐VCH, Weinheim, Germany. | |
| Machius, M., Declerck, N., Huber, R., and Wiegand, G. 2003. Kinetic stabilization of Bacillus licheniformis alpha‐amylase through introduction of hydrophobic residues at the surface. J. Biol. Chem. 278:11,546‐11,553. | |
| Magnusson, A.O., Takwa, M., Hamberg, A., and Hult, K. 2005. An S‐selective lipase was created by rational redesign and the enantioselectivity increased with temperature. Angew. Chem. Int. Ed. 44:4582‐4585. | |
| Matthews, B.W., Nicholson, H., and Becktel, W.J. 1987. Enhanced protein thermostability from site‐directed mutations that decrease the entropy of unfolding. Proc. Natl. Acad. Sci. U.S.A. 84:6663‐6667. | |
| McCarthy, A. 2003. Metabolix, Inc. and Tepha, Inc. Bioplastics for industry and medical devices. Chem. Biol. 10:893‐894. | |
| Moore, J.C. and Arnold, F.H. 1996. Directed evolution of a para‐nitrobenzyl esterase for aqueous‐organic solvents. Nat. Biotechnol. 14:458‐467. | |
| Morley, K.L. and Kazlauskas, R.J. 2005. Improving enzyme properties: When are closer mutations better? Trends Biotechnol. 23:231‐237. | |
| 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. | |
| Otten, L.G. and Quax, W.J. 2005. Directed evolution: Selecting today's biocatalysts. Biomol. Eng. 22:1‐9. | |
| Padhi, S.K., Fujii, R., Legatt, G.A., Fossum, S.L., Berchtold, R., and Kazlauskas, R.J. 2010. Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions. Chem. Biol. 17:863‐871. | |
| Park, S., Morley, K.L., Horsman, G.P., Holmquist, M., Hult, K., and Kazlauskas, R.J. 2005. Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations. Chem. Biol. 12:45‐54. | |
| Qian, Z. and Lutz, S. 2005. Improving the catalytic activity of Candida antarctica lipase B by circular permutation. J. Am. Chem. Soc. 127:13466‐13467. | |
| Raillard, S., Krebber, A., Chen, Y., Ness, J.E., Bermudez, E., Trinidad, R., Fullem, R., Davis, C., Welch, M., Seffernick, J., Wackett, L.P., Stemmer, W.P., and Minshull, J. 2001. Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. Chem. Biol. 8:891‐898. | |
| Reetz, M.T. and Sanchis, J. 2008. Constructing and analyzing the fitness landscape of an experimental evolutionary process. ChemBioChem 9:2260‐2267. | |
| Reetz, M.T. and Wu, S. 2008. Greatly reduced amino acid alphabets in directed evolution: making the right choice for saturation mutagenesis at homologous enzyme positions. Chem. Commun. 43:5499‐5501. | |
| Reetz, M.T., Zonta, A., Schimossek, K., Liebeton, K., and Jaeger, K.‐E. 1997. Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew. Chem. Int. Ed. Engl. 36:2830‐2832. | |
| Reetz, M.T., Torre, C., Eipper, A., Lohmer, R., Hermes, M., Brunner, B., Maichele, A., Bocola, M., Arand, M., Cronin, A., Genzel, Y., Archelas, A., and Furstoss, R. 2004. Enhancing the enantioselectivity of an epoxide hydrolase by directed evolution. Org. Lett. 6:177‐180. | |
| Reetz, M.T., Bocola, M., Carballeira, J.D., Zha, D.X., and Vogel, A. 2005. Expanding the range of substrate acceptance of enzymes: Combinatorial active‐site saturation test. Angew. Chem. Int. Ed. 44:4192‐4196. | |
| Reetz, M.T., Carballeira, J.D., and Vogel, A. 2006a. Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew. Chem. Int. Ed. 45:7745‐7751. | |
| Reetz, M.T., Wang, L.W., and Bocola, M. 2006b. Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein‐sequence space. Angew. Chem. Int. Ed. 45:1236‐1241. | |
| Reetz, M.T., Kahakeaw, D., and Lohmer, R. 2008. Addressing the numbers problem in directed evolution. ChemBioChem 9:1797‐1804. | |
| Reetz, M.T., Soni, P., Fernandez, L., Gumulya, Y., and Carballeira, J.D. 2010. Increasing the stability of an enzyme toward hostile organic solvents by directed evolution based on iterative saturation mutagenesis using the B‐FIT method. Chem. Commun. 46:8657‐8658. | |
| Reymond, J.L., ed. 2005. Enzyme Assays. Wiley‐VCH, Weinheim, Germany. | |
| Röthlisberger, D., Khersonsky, O., Wollacott, A.M., Jiang, L., DeChancie, J., Betker, J., Gallaher, J.L., Althoff, E.A., Zanghellini, A., Dym, O., Albeck, S., Houk, K.N., Tawfik, D.S., and Baker, D. 2008. Kemp elimination catalysts by computational enzyme design. Nature 453:190‐195. | |
| Savile, C.K., Janey, J.M., Mundorff, E.C., Moore, J.C., Tam, S., Jarvis, W.R., Colbeck, J.C., Krebber, A., Fleitz, F.J., Brands, J., Devine, P.N., Huisman, G.W., and Hughes, G.J. 2010. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329:305‐309. | |
| Schmidt, M., Hasenpusch, D., Kähler, M., Kirchner, U., Wiggenhorn, K., Langel, W., and Bornscheuer, U.T. 2006. Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block. ChemBioChem 7:805‐809. | |
| Schmidt‐Dannert, C., Umeno, D., and Arnold, F.H. 2000. Molecular breeding of carotenoid biosynthetic pathways. Nat. Biotechnol. 18:750‐753. | |
| Seebeck, F.P. and Hilvert, D. 2003. Conversion of a PLP‐dependent racemase into an aldolase by a single active site mutation. J. Am. Chem. Soc. 125:10,158‐10,159. | |
| Seelig, B. and Szostak, J.W. 2007. Selection and evolution of enzymes from a partially randomized non‐catalytic scaffold. Nature 448:828‐831. | |
| Siegel, J.B., Zanghellini, A., Lovick, H.M., Kiss, G., Lambert, A.R., St. Clair, J.L., Gallaher, J.L., Hilvert, D., Gelb, M.H., Stoddard, B.L., Houk, K.N., Michael, F.E., and Baker, D. 2010. Computational design of an enzyme catalyst for a stereoselective bimolecular Diels‐Alder reaction. Science 329:309‐313. | |
| Stemmer, W.P.C. 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389‐391. | |
| Suzuki, Y. 1999. The proline rule. A strategy for protein thermal stabilization. Proc. Jpn. Acd. Ser. B: Phys. Biol. Sci. 75:133‐137. | |
| Terao, Y., Miyamoto, K., and Ohta, H. 2006. Introduction of single mutation changes arylmalonate decarboxylase to racemase. Chem. Commun. 34:3600‐3602. | |
| Terao, Y., Ijima, Y., Miyamoto, K., and Ohta, H. 2007. Inversion of enantioselectivity of arylmalonate decarboxylase via site‐directed mutation based on the proposed reaction mechanism. J. Mol. Catal. B: Enzym. 45:15‐20. | |
| Van den Burg, B., Vriend, G., Veltman, O.R., Venema, G., and Eijsink, V.G. 1998. Engineering an enzyme to resist boiling. Proc. Natl. Acad. Sci. U.S.A. 95:2056‐2060. | |
| Vazquez‐Figueroa, E., Yeh, V., Broering, J.M., Chaparro‐Riggers, J.F., and Bommarius, A.S. 2008. Thermostable variants constructed via the structure‐guided consensus method also show increased stability in salts solutions and homogeneous aqueous‐organic media. Prot. Eng. Des. Sel. 21:673‐680. | |
| Vick J.E., Schmidt, D.M.Z., and Gerlt, J.A. 2005. Evolutionary potential of (beta/alpha)8‐barrels: In vitro enhancement of a “new” reaction in the enolase superfamily. Biochemistry 44:11,722‐11,729. | |
| Weinreich, D.M., Delaney, N.F., Depristo, M.A., and Hartl, D.L. 2006. Darwinian evolution can follow only very few mutational paths to fitter proteins. Science 312:111‐114. | |
| Whittle, E. and Shanklin, J. 2001. Engineering delta 9‐16:0‐acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9‐18:0‐ACP desaturase. J. Biol. Chem. 276:21,500‐21,005. | |
| Wilson, D.S. and Keefe, A.D. 2000. Random mutagenesis by PCR. Curr. Protoc. Mol. Biol. 51:8.3.1‐8.3.9. | |
| Wong, T.S., Roccatano, D., Zacharias, M., and Schwaneberg, U. 2006. A statistical analysis of random mutagenesis methods used for directed protein evolution. J. Mol. Biol. 355:858‐871. | |
| Xiang, H., Luo, L., Taylor, K.L., and Dunaway‐Mariano, D. 1999. Interchange of catalytic activity within the 2‐enoyl‐coenzyme A hydratase/isomerase superfamily based on a common active site template. Biochemistry 38:7638‐7652. | |
| Xiao, Z., Bergeron, H., Grosse, S., Beauchemin, M., Garron, M.L., Shaya, D., Sulea, T., Cygler, M., and Lau, P.C. 2008. Improvement of the thermostability and activity of a pectate lyase by single amino acid substitutions, using a strategy based on melting‐temperature‐guided sequence alignment. Appl. Environ. Microbiol. 74:1183‐1189. | |
| Zha, D.X., Wilensek, S., Hermes, M., Jaeger, K.E., and Reetz, M.T. 2001. Complete reversal of enantioselectivity of an enzyme‐catalyzed reaction by directed evolution. Chem. Commun. 24:2664‐2665. | |
| Key References | |
| Lutz and Bornscheuer, 2009. See above. | |
| This two volume edited book comprehensively covers current protein engineering methods. |
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
