Cellular and Molecular Life Sciences

, Volume 68, Issue 7, pp 1207–1214 | Cite as

Technologies of directed protein evolution in vivo

  • Artem Blagodatski
  • Vladimir L. KatanaevEmail author


Directed evolution of proteins for improved or modified functionality is an important branch of modern biotechnology. It has traditionally been performed using various in vitro methods, but more recently, methods of in vivo artificial evolution come into play. In this review, we discuss and compare prokaryotic and eukaryotic-based systems of directed protein evolution in vivo, highlighting their benefits and current limitations and focusing on the biotechnological potential of vertebrate immune cells for the generation of protein diversity by means of the immunoglobulin diversification machinery.


Directed evolution Protein engineering Mutator strain Cell culture-based systems Somatic hypermutation Gene conversion 



Activation-induced deaminase


Blue fluorescent protein


Enhanced GFP


Fluorescence-activated cell sorting


Green fluorescent protein


G protein-coupled receptor


Isopropyl β-d-1-thiogalactopyranoside


Monomeric red fluorescent protein


Polymerase chain reaction


Trichostatin A



The work was supported by grant No. 02.740.11.5016 from the Russian Ministry of Science and by Deutsche Forschungsgemeinschaft (TR SFB-11) to V.L.K. A.B. is a recipient of the Russian state scholarship No. 8084 p/12612 “Participant of the Youth Contest for Science and Innovation”.


  1. 1.
    Kaur J, Sharma R (2006) Directed evolution: an approach to engineer enzymes. Crit Rev Biotechnol 26(3):165–199. doi: 10.1080/07388550600851423 PubMedCrossRefGoogle Scholar
  2. 2.
    Jackel C, Kast P, Hilvert D (2008) Protein design by directed evolution. Annu Rev Biophys 37:153–173. doi: 10.1146/annurev.biophys.37.032807.125832 PubMedCrossRefGoogle Scholar
  3. 3.
    Bottcher D, Bornscheuer UT Protein engineering of microbial enzymes. Curr Opin Microbiol 13 (3):274–282. S1369-5274(10)00015-9 [pii]Google Scholar
  4. 4.
    Dougherty MJ, Arnold FH (2009) Directed evolution: new parts and optimized function. Curr Opin Biotechnol 20(4):486–491. doi: 10.1016/j.copbio.2009.08.005 PubMedCrossRefGoogle Scholar
  5. 5.
    Stemmer WP (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370(6488):389–391. doi: 10.1038/370389a0 PubMedCrossRefGoogle Scholar
  6. 6.
    Ness JE, Welch M, Giver L, Bueno M, Cherry JR, Borchert TV, Stemmer WP, Minshull J (1999) DNA shuffling of subgenomic sequences of subtilisin. Nat Biotechnol 17(9):893–896. doi: 10.1038/12884 PubMedCrossRefGoogle Scholar
  7. 7.
    Harayama S (1998) Artificial evolution by DNA shuffling. Trends Biotechnol 16(2):76–82. S0167-7799(97)01158-X [pii]PubMedCrossRefGoogle Scholar
  8. 8.
    Shen B (2002) PCR approaches to DNA mutagenesis and recombination. An overview. Methods Mol Biol 192:167–174. doi: 10.1385/1-59259-177-9:167 PubMedGoogle Scholar
  9. 9.
    Matsuura T, Yomo T (2006) In vitro evolution of proteins. J Biosci Bioeng 101(6):449–456. doi: 10.1263/jbb.101.449 PubMedCrossRefGoogle Scholar
  10. 10.
    Lipovsek D, Pluckthun A (2004) In vitro protein evolution by ribosome display and mRNA display. J Immunol Methods 290(1–2):51–67. doi: 10.1016/j.jim.2004.04.008 PubMedCrossRefGoogle Scholar
  11. 11.
    Smith GP, Petrenko VA (1997) Phage display. Chem Rev 97(2):391–410PubMedCrossRefGoogle Scholar
  12. 12.
    Labrou NE (2010) Random mutagenesis methods for in vitro directed enzyme evolution. Curr Protein Pept Sci 11(1):91–100Google Scholar
  13. 13.
    Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91(22):10747–10751PubMedCrossRefGoogle Scholar
  14. 14.
    Cirino PC, Mayer KM, Umeno D (2003) Generating mutant libraries using error-prone PCR. Methods Mol Biol 231:3–9. doi: 10.1385/1-59259-395-X:3 PubMedGoogle Scholar
  15. 15.
    Sarkar CA, Dodevski I, Kenig M, Dudli S, Mohr A, Hermans E, Pluckthun A (2008) Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proc Natl Acad Sci USA 105(39):14808–14813. doi: 10.1073/pnas.0803103105 PubMedCrossRefGoogle Scholar
  16. 16.
    Liu R, Barrick JE, Szostak JW, Roberts RW (2000) Optimized synthesis of RNA-protein fusions for in vitro protein selection. Methods Enzymol 318:268–293PubMedCrossRefGoogle Scholar
  17. 17.
    Hanes J, Pluckthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci USA 94(10):4937–4942PubMedCrossRefGoogle Scholar
  18. 18.
    Spirin AS (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 22(10):538–545. doi: 10.1016/j.tibtech.2004.08.012 PubMedCrossRefGoogle Scholar
  19. 19.
    Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. Methods Mol Biol 57:375–385. doi: 10.1385/0-89603-332-5:375 PubMedGoogle Scholar
  20. 20.
    Chusacultanachai S, Yuthavong Y (2004) Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments. Methods Mol Biol 270:319–334. doi: 10.1385/1-59259-793-9:319 PubMedGoogle Scholar
  21. 21.
    Bornscheuer UT, Altenbuchner J, Meyer HH (1998) Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones. Biotechnol Bioeng 58(5):554–559. doi: 10.1002/(SICI)1097-0290(19980605)58:5<554::AID-BIT12>3.0.CO;2-B PubMedCrossRefGoogle Scholar
  22. 22.
    Bornscheuer UT, Altenbuchner J, Meyer HH (1999) Directed evolution of an esterase: screening of enzyme libraries based on pH indicators and a growth assay. Bioorg Med Chem 7(10):2169–2173. S0968-0896(99)00147-9 [pii]PubMedCrossRefGoogle Scholar
  23. 23.
    Callanan MJ, Russell WM, Klaenhammer TR (2007) Modification of Lactobacillus beta-glucuronidase activity by random mutagenesis. Gene 389(2):122–127. doi: 10.1016/j.gene.2006.10.022 PubMedCrossRefGoogle Scholar
  24. 24.
    Lu X, Hirata H, Yamaji Y, Ugaki M, Namba S (2001) Random mutagenesis in a plant viral genome using a DNA repair-deficient mutator Escherichia coli strain. J Virol Methods 94(1–2):37–43. S0166093401002701 [pii]PubMedCrossRefGoogle Scholar
  25. 25.
    Nakashima N, Tamura T (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli. Nucleic Acids Res 37(15):e103. doi: 10.1093/nar/gkp498 PubMedCrossRefGoogle Scholar
  26. 26.
    Yang H, Miller JH (2008) Deletion of dnaN1 generates a mutator phenotype in Bacillus anthracis. DNA Repair (Amst) 7(3):507–514. doi: 10.1016/j.dnarep.2007.10.003 CrossRefGoogle Scholar
  27. 27.
    Camps M, Naukkarinen J, Johnson BP, Loeb LA (2003) Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I. Proc Natl Acad Sci USA 100(17):9727–9732. doi: 10.1073/pnas.1333928100 PubMedCrossRefGoogle Scholar
  28. 28.
    Itoh T, Tomizawa J (1979) Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H, and DNA polymerase I. Cold Spring Harb Symp Quant Biol 43(Pt 1):409–417PubMedGoogle Scholar
  29. 29.
    Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460(7257):894–898. doi: 10.1038/nature08187 PubMedCrossRefGoogle Scholar
  30. 30.
    Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci USA 98(12):6742–6746. doi: 10.1073/pnas.121164898 PubMedCrossRefGoogle Scholar
  31. 31.
    Pirakitikulr N, Ostrov N, Peralta-Yahya P, Cornish VW PCR less library mutagenesis via oligonucleotide recombination in yeast. Protein Sci 19(12):2336–2346. doi: 10.1002/pro.513
  32. 32.
    Weigert MG, Cesari IM, Yonkovich SJ, Cohn M (1970) Variability in the lambda light chain sequences of mouse antibody. Nature 228(5276):1045–1047PubMedCrossRefGoogle Scholar
  33. 33.
    Kim S, Davis M, Sinn E, Patten P, Hood L (1981) Antibody diversity: somatic hypermutation of rearranged VH genes. Cell 27(3 Pt 2):573–581. doi: 0092-8674(81)90399-8 PubMedCrossRefGoogle Scholar
  34. 34.
    McKean D, Huppi K, Bell M, Staudt L, Gerhard W, Weigert M (1984) Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. Proc Natl Acad Sci USA 81(10):3180–3184PubMedCrossRefGoogle Scholar
  35. 35.
    Milstein C, Rada C (1995) The maturation of the antibody response. In: Honjo T, Alt F (eds) Immunoglobulin genes. Academic Press Limited, London, pp 57–83CrossRefGoogle Scholar
  36. 36.
    Papavasiliou FN, Schatz DG (2002) Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell 109(Suppl):S35–S44PubMedCrossRefGoogle Scholar
  37. 37.
    Martin A, Scharff MD (2002) AID and mismatch repair in antibody diversification. Nat Rev Immunol 2(8):605–614. doi: 10.1038/nri858 PubMedGoogle Scholar
  38. 38.
    Neuberger MS, Harris RS, Di Noia J, Petersen-Mahrt SK (2003) Immunity through DNA deamination. Trends Biochem Sci 28(6):305–312. S0968000403001117 [pii]PubMedCrossRefGoogle Scholar
  39. 39.
    Rajewsky K, Forster I, Cumano A (1987) Evolutionary and somatic selection of the antibody repertoire in the mouse. Science 238(4830):1088–1094PubMedCrossRefGoogle Scholar
  40. 40.
    Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102(5):553–563. S0092-8674(00)00078-7 [pii]PubMedCrossRefGoogle Scholar
  41. 41.
    Bransteitter R, Pham P, Scharff MD, Goodman MF (2003) Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc Natl Acad Sci USA 100(7):4102–4107. doi: 10.1073/pnas.0730835100 PubMedCrossRefGoogle Scholar
  42. 42.
    Pham P, Bransteitter R, Petruska J, Goodman MF (2003) Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424(6944):103–107. doi: 10.1038/nature01760 PubMedCrossRefGoogle Scholar
  43. 43.
    Peters A, Storb U (1996) Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4(1):57–65. doi: S1074-7613(00)80298-8 PubMedCrossRefGoogle Scholar
  44. 44.
    Cascalho M, Wong J, Steinberg C, Wabl M (1998) Mismatch repair co-opted by hypermutation. Science 279(5354):1207–1210PubMedCrossRefGoogle Scholar
  45. 45.
    Di Noia J, Neuberger MS (2002) Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419(6902):43–48. doi: 10.1038/nature00981 PubMedCrossRefGoogle Scholar
  46. 46.
    Petersen-Mahrt SK, Harris RS, Neuberger MS (2002) AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418(6893):99–103. doi: 10.1038/nature00862 PubMedCrossRefGoogle Scholar
  47. 47.
    Yelamos J, Klix N, Goyenechea B, Lozano F, Chui YL, Gonzalez Fernandez A, Pannell R, Neuberger MS, Milstein C (1995) Targeting of non-Ig sequences in place of the V segment by somatic hypermutation. Nature 376(6537):225–229. doi: 10.1038/376225a0 PubMedCrossRefGoogle Scholar
  48. 48.
    Bachl J, Carlson C, Gray-Schopfer V, Dessing M, Olsson C (2001) Increased transcription levels induce higher mutation rates in a hypermutating cell line. J Immunol 166(8):5051–5057PubMedGoogle Scholar
  49. 49.
    Yoshikawa K, Okazaki IM, Eto T, Kinoshita K, Muramatsu M, Nagaoka H, Honjo T (2002) AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts. Science 296(5575):2033–2036. doi: 10.1126/science.1071556 PubMedCrossRefGoogle Scholar
  50. 50.
    Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99(12):7877–7882. doi: 10.1073/pnas.082243699;99/12/7877 PubMedCrossRefGoogle Scholar
  51. 51.
    Sale JE, Neuberger MS (1998) TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line. Immunity 9(6):859–869. S1074-7613(00)80651-2 [pii]PubMedCrossRefGoogle Scholar
  52. 52.
    Wang L, Jackson WC, Steinbach PA, Tsien RY (2004) Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc Natl Acad Sci USA 101(48):16745–16749. doi: 10.1073/pnas.0407752101 PubMedCrossRefGoogle Scholar
  53. 53.
    Arakawa H, Hauschild J, Buerstedde JM (2002) Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295(5558):1301–1306. doi: 10.1126/science.1067308 PubMedCrossRefGoogle Scholar
  54. 54.
    Arakawa H, Saribasak H, Buerstedde JM (2004) Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate. PLoS Biol 2(7):E179. doi: 10.1371/journal.pbio.0020179 PubMedCrossRefGoogle Scholar
  55. 55.
    Buerstedde JM, Takeda S (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67(1):179–188. doi: 0092-8674(91)90581-I PubMedCrossRefGoogle Scholar
  56. 56.
    Seo H, Masuoka M, Murofushi H, Takeda S, Shibata T, Ohta K (2005) Rapid generation of specific antibodies by enhanced homologous recombination. Nat Biotechnol 23(6):731–735. doi: 10.1038/nbt1092 PubMedCrossRefGoogle Scholar
  57. 57.
    Seo H, Hashimoto S, Tsuchiya K, Lin W, Shibata T, Ohta K (2006) An ex vivo method for rapid generation of monoclonal antibodies (ADLib system). Nat Protoc 1(3):1502–1506. doi: 10.1038/nprot.2006.248 PubMedCrossRefGoogle Scholar
  58. 58.
    Seo H, Yamada T, Hashimoto S, Lin W, Ohta K (2007) Modulation of immunoglobulin gene conversion in chicken DT40 by enhancing histone acetylation, and its application to antibody engineering. Biotechnol Genet Eng Rev 24:179–193PubMedGoogle Scholar
  59. 59.
    Kanayama N, Todo K, Takahashi S, Magari M, Ohmori H (2006) Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line. Nucleic Acids Res 34(2):e10. 34/2/e10 [pii]PubMedCrossRefGoogle Scholar
  60. 60.
    Arakawa H, Kudo H, Batrak V, Caldwell RB, Rieger MA, Ellwart JW, Buerstedde JM (2008) Protein evolution by hypermutation and selection in the B cell line DT40. Nucleic Acids Res 36(1):e1. doi: 10.1093/nar/gkm616 PubMedCrossRefGoogle Scholar
  61. 61.
    Arakawa H, Lodygin D, Buerstedde JM (2001) Mutant loxP vectors for selectable marker recycle and conditional knock-outs. BMC Biotechnol 1:7PubMedCrossRefGoogle Scholar
  62. 62.
    Betz AG, Milstein C, Gonzalez-Fernandez A, Pannell R, Larson T, Neuberger MS (1994) Elements regulating somatic hypermutation of an immunoglobulin kappa gene: critical role for the intron enhancer/matrix attachment region. Cell 77(2):239–248. 0092-8674(94)90316-6 [pii]PubMedCrossRefGoogle Scholar
  63. 63.
    Odegard VH, Schatz DG (2006) Targeting of somatic hypermutation. Nat Rev Immunol 6(8):573–583. doi: 10.1038/nri1896 PubMedCrossRefGoogle Scholar
  64. 64.
    Kothapalli N, Norton DD, Fugmann SD (2008) Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus. J Immunol 180(4):2019–2023. 180/4/2019 [pii]PubMedGoogle Scholar
  65. 65.
    Blagodatski A, Batrak V, Schmidl S, Schoetz U, Caldwell RB, Arakawa H, Buerstedde JM (2009) A cis-acting diversification activator both necessary and sufficient for AID-mediated hypermutation. PLoS Genet 5(1):e1000332. doi: 10.1371/journal.pgen.1000332 PubMedCrossRefGoogle Scholar
  66. 66.
    Arakawa H, Buerstedde JM (2009) Activation-induced cytidine deaminase-mediated hypermutation in the DT40 cell line. Philos Trans R Soc Lond B Biol Sci 364(1517):639–644. doi: 10.1098/rstb.2008.0202 PubMedCrossRefGoogle Scholar

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© Springer Basel AG 2010

Authors and Affiliations

  1. 1.Institute of Protein ResearchRussian Academy of SciencesPushchinoRussian Federation
  2. 2.University of KonstanzKonstanzGermany

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