Skip to main content

Protein Engineering: Past, Present, and Future

  • Protocol
  • First Online:
Protein Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1685))

Abstract

The last decade has seen a dramatic increase in the utilization of enzymes as green and sustainable (bio)catalysts in pharmaceutical and industrial applications. This trend has to a significant degree been fueled by advances in scientists’ and engineers’ ability to customize native enzymes by protein engineering. A review of the literature quickly reveals the tremendous success of this approach; protein engineering has generated enzyme variants with improved catalytic activity, broadened or altered substrate specificity, as well as raised or reversed stereoselectivity. Enzymes have been tailored to retain activity at elevated temperatures and to function in the presence of organic solvents, salts and pH values far from physiological conditions. However, readers unfamiliar with the field will soon encounter the confusingly large number of experimental techniques that have been employed to accomplish these engineering feats. Herein, we use history to guide a brief overview of the major strategies for protein engineering—past, present, and future.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Institutional subscriptions

References

  1. Rosenthaler L (1908) Durch Enzyme bewirkte asymmetrische Synthese. Biochem Z 14:238–253

    CAS  Google Scholar 

  2. Winter G et al (1982) Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature 299:756–758

    Article  CAS  PubMed  Google Scholar 

  3. Wilkinson AJ et al (1983) Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry 22:3581–3586

    Article  CAS  PubMed  Google Scholar 

  4. Estell DA, Graycar TP, Wells JA (1985) Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. J Biol Chem 260:6518–6521

    CAS  PubMed  Google Scholar 

  5. Gorontzy T et al (1994) Microbial degradation of explosives and related compounds. Crit Rev Microbiol 20:265–284

    Article  CAS  PubMed  Google Scholar 

  6. Singh BK, Walker A (2006) Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev 30:428–471

    Article  CAS  PubMed  Google Scholar 

  7. Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74:417–433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Iredell J, Brown J, Tagg K (2016) Antibiotic resistance in Enterobacteriaceae: mechanisms and clinical implications. BMJ 352:h6420

    Article  PubMed  Google Scholar 

  9. Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. Genome Res 2:28–33

    Article  CAS  Google Scholar 

  10. Firth AE, Patrick WM (2005) Statistics of protein library construction. Bioinformatics 21:3314–3315

    Article  CAS  PubMed  Google Scholar 

  11. Arnold FH (1990) Engineering enzymes for non-aqueous solvents. Trends Biotechnol 8:244–249

    Article  CAS  PubMed  Google Scholar 

  12. Dube DK et al (1991) Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene. Biochemistry 30:11760–11767

    Article  CAS  PubMed  Google Scholar 

  13. Chen KQ, Arnold FH (1993) Tuning the activity of an enzyme for unusual environments–sequential random mutagenesis of subtilisin-E for catalysis in dimethylformamide. Proc Nat Acad Sci USA 90:5618–5622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Moore JC, Arnold FH (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat Biotechnol 14:458–467

    Article  CAS  PubMed  Google Scholar 

  15. Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci U S A 91:10747–10751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stemmer WP (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389–391

    Article  CAS  PubMed  Google Scholar 

  17. Zhao H, Giver L, Shao Z et al (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16:258–261

    Article  CAS  PubMed  Google Scholar 

  18. MĂĽller KM, Stebel SC, Knall S et al (2005) Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution. Nucleic Acids Res 33:e117

    Article  PubMed  PubMed Central  Google Scholar 

  19. Crameri A, Raillard SA, Bermudez E et al (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391:288–291

    Article  CAS  PubMed  Google Scholar 

  20. Ness JE, Welch M, Giver L et al (1999) DNA shuffling of subgenomic sequences of subtilisin. Nat Biotechnol 17:893–896

    Article  CAS  PubMed  Google Scholar 

  21. Kolkman JA, Stemmer WP (2001) Directed evolution of proteins by exon shuffling. Nat Biotechnol 19:423–428

    Article  CAS  PubMed  Google Scholar 

  22. Patnaik R, Louie S, Gavrilovic V et al (2002) Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol 20:707–712

    Article  CAS  PubMed  Google Scholar 

  23. Romero PA, Arnold FA (2009) Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 10:866–876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Patrick WM, Firth AE, Blackburn JM (2003) User-friendly algorithms for estimating completeness and diversity in randomized protein-encoding libraries. Protein Eng 16:451–457

    Article  CAS  PubMed  Google Scholar 

  25. Acevedo-Rocha CG, Agudo R, Reetz MT (2014) Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems. J Biotechnol 191:3–10

    Article  CAS  PubMed  Google Scholar 

  26. Martis EA, Badve RR (2011) High-throughput screening: the hits and leads of drug discovery–an overview. J Appl Pharm Sci 1:2–10

    Google Scholar 

  27. Lutz S (2010) Beyond directed evolution–semi-rational protein engineering and design. Curr Opin Biotechnol 21:734–743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Clouthier CM, Kayser MM, Reetz MT (2006) Designing new Baeyer-Villiger monooxygenases using restricted CASTing. J Org Chem 71:8431–8437

    Article  CAS  PubMed  Google Scholar 

  29. Reetz MT, Carballeira JD, Peyralans J et al (2006) Expanding the substrate scope of enzymes: combining mutations obtained by CASTing. Chem Eur J 12:6031–6038

    Article  CAS  PubMed  Google Scholar 

  30. Reetz MT, Wang LW, Bocola M (2006) Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed 45:1236–1241

    Article  CAS  Google Scholar 

  31. Agudo R, Roiban GD, Reetz MT (2012) Achieving regio- and enantioselectivity of P450-catalyzed oxidative CH activation of small functionalized molecules by structure-guided directed evolution. ChemBioChem 13:1465–1473

    Article  CAS  PubMed  Google Scholar 

  32. Gumulya Y, Sanchis J, Reetz MT (2012) Many pathways in laboratory evolution can lead to improved enzymes: how to escape from local minima. ChemBioChem 13:1060–1066

    Article  CAS  PubMed  Google Scholar 

  33. Parra LP, Agudo R, Reetz MT (2013) Directed evolution by using iterative saturation mutagenesis based on multiresidue sites. ChemBioChem 14:2301–2309

    Article  CAS  PubMed  Google Scholar 

  34. Kourist R, Jochens H, Bartsch S et al (2010) The α/β-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering. ChemBioChem 11:1635–1643

    Article  CAS  PubMed  Google Scholar 

  35. Kuipers RK, Joosten HJ, van Berkel WJ et al (2010) 3DM: systematic analysis of heterogeneous superfamily data to discover protein functionalities. Proteins 78:2101–2113

    CAS  PubMed  Google Scholar 

  36. Krieger E, Koraimann G, Vriend G (2002) Increasing the precision of comparative models with YASARA NOVA–a self-parameterizing force field. Proteins 47:393–402

    Article  CAS  PubMed  Google Scholar 

  37. Das R, Baker D (2008) Macromolecular modeling with Rosetta. Annu Rev Biochem 77:363–382

    Article  CAS  PubMed  Google Scholar 

  38. Richter F, Leaver-Fay A, Khare SD et al (2011) De novo enzyme design using Rosetta3. PLoS One 6:e19230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bartsch S, Wybenga GG, Jansen M et al (2013) Redesign of a phenylalanine aminomutase into a phenylalanine ammonia lyase. ChemCatChem 5:1797–1802

    Article  CAS  Google Scholar 

  40. Floor RJ, Wijma HJ, Colpa DI et al (2014) Computational library design for increasing haloalkane dehalogenase stability. ChemBioChem 15:1660–1672

    Article  CAS  PubMed  Google Scholar 

  41. Wijma HJ, Floor HJ, Jekel PA et al (2014) Computationally designed libraries for rapid enzyme stabilization. Protein Eng Des Sel 27:49–58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Liao J, Warmuth MK, Govindarajan S et al (2007) Engineering proteinase K using machine learning and synthetic genes. BMC Biotechnol 7:16

    Article  PubMed  PubMed Central  Google Scholar 

  43. Ehren J, Govindarajan S, Morón B et al (2008) Protein engineering of improved prolyl endopeptidases for celiac sprue therapy. Protein Eng Des Sel 21:699–707

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Midelfort KS, Kumar R, Han S et al (2013) Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin. Protein Eng Des Sel 26:25–33

    Article  CAS  PubMed  Google Scholar 

  45. Govindarajan S, Mannervik B, Silverman JA et al (2015) Mapping of amino acid substitutions conferring herbicide resistance in wheat glutathione transferase. ACS Synth Biol 4:221–227

    Article  CAS  PubMed  Google Scholar 

  46. Lutz S, Patrick WM (2004) Novel methods for directed evolution of enzymes: quality, not quantity. Curr Opin Biotechnol 15:291–297

    Article  CAS  PubMed  Google Scholar 

  47. Lodish H, Berk A, Zipursky SL et al (2000) Molecular cell biology, 4th edn. W. H. Freeman, New York, Section 7.1, DNA cloning with plasmid vectors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21498/

  48. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557

    Article  CAS  PubMed  Google Scholar 

  49. Chao G, Lau WL, Hackel BJ et al (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protocols 1:755–768

    Article  CAS  PubMed  Google Scholar 

  50. Bratkovič T (2009) Progress in phage display: evolution of the technique and its applications. Cell Mol Life Sci 67:749–767

    Article  Google Scholar 

  51. Çelik E, Fischer AC, Guarino C et al (2010) A filamentous phage display system for N-linked glycoproteins. Protein Sci 19:2006–2013

    Article  PubMed  PubMed Central  Google Scholar 

  52. Karlsson AJ, Lim HK, Xu H et al (2012) Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins. J Mol Biol 416:94–107

    Article  CAS  PubMed  Google Scholar 

  53. Tawfik DS, Griffiths AD (1998) Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16:652–656

    Article  CAS  PubMed  Google Scholar 

  54. Bernath K, Hai M, Mastrobattista E et al (2004) In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting. Anal Biochem 325:151–157

    Article  CAS  PubMed  Google Scholar 

  55. Aharoni A, Griffiths AD, Tawfik DS (2005) High-throughput screens and selections of enzyme-encoding genes. Curr Opin Chem Biol 9:210–216

    Article  CAS  PubMed  Google Scholar 

  56. Agresti JJ, Antipov E, Abate AR et al (2010) Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc Natl Acad Sci U S A 107:4004–4009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Fischlechner M, Shaerli Y, Mohamed MF et al (2014) Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat Chem 6:791–796

    Article  CAS  PubMed  Google Scholar 

  58. Ostafe R, Prodanovic R, Nazor J et al (2014) Ultra-high-throughput screening method for the directed evolution of glucose oxidase. Chem Biol 21:414–421

    Article  CAS  PubMed  Google Scholar 

  59. Zinchenko A, Devenish SRA, Kintses B et al (2014) One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution. Anal Chem 86:2526–2533

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Romero PA, Tran TM, Abate AR (2015) Dissecting enzyme function with microfluidic-based deep mutational scanning. Proc Natl Acad Sci U S A 112:7159–7164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Bornscheuer UT, Huisman GW, Kazlauskas RJ et al (2012) Engineering the third wave of biocatalysis. Nature 485:185–194

    Article  CAS  PubMed  Google Scholar 

  62. Prier CK, Arnold FH (2015) Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts. J Am Chem Soc 137:13992–14006

    Article  CAS  PubMed  Google Scholar 

  63. Coelho PS, Brustad EM, Kannan A et al (2013) Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339:307–310

    Article  CAS  PubMed  Google Scholar 

  64. Bordeaux M, Tyagi V, Fasan R (2015) Highly diastereoselective and enantioselective olefin cyclopropanation using engineered myoglobin-based catalysts. Angew Chem Int Ed 54:1744–1748

    Article  CAS  Google Scholar 

  65. Heinisch T, Pellizzoni M, Dürrenberger M et al (2015) Improving the catalytic performance of an artificial metalloenzyme by computational design. J Am Chem Soc 137:10414–10419

    Article  CAS  PubMed  Google Scholar 

  66. Srivastava P, Yang H, Ellis-Guardiola K et al (2015) Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation. Nat Commun 6:7789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Althoff EA, Wang L, Jiang L et al (2012) Robust design and optimization of retroaldol enzymes. Protein Sci 21:717–726

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Giger L, Caner S, Obexer R et al (2013) Evolution of a designed retro-aldolase leads to complete active site remodeling. Nat Chem Biol 9:494–498

    Google Scholar 

Download references

Acknowledgments

We thank the members of the Lutz lab for helpful comments and suggestions on the manuscript. Financial support in part by the US National Science Foundation (CBET-1159434 & CBET-1546790) is gratefully acknowledged.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stefan Lutz .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lutz, S., Iamurri, S.M. (2018). Protein Engineering: Past, Present, and Future. In: Bornscheuer, U., Höhne, M. (eds) Protein Engineering. Methods in Molecular Biology, vol 1685. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7366-8_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7366-8_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7364-4

  • Online ISBN: 978-1-4939-7366-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics