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Directed evolution of proteins by exon shuffling

Abstract

Evolution of eukaryotes is mediated by sexual recombination of parental genomes. Crossovers occur in random, but homologous, positions at a frequency that depends on DNA length. As exons occupy only 1% of the human genome and introns about 24%, by far most of the crossovers occur between exons, rather than inside. The natural process of creating new combinations of exons by intronic recombination is called exon shuffling. Our group is developing in vitro formats for exon shuffling and applying these to the directed evolution of proteins. Based on the splice frame junctions, nine classes of exons and three classes of introns can be distinguished. Splice frame diagrams of natural genes show how the splice frame rules govern exon shuffling. Here, we review various approaches to constructing libraries of exon-shuffled genes. For example, exon shuffling of human pharmaceutical proteins can generate libraries in which all of the sequences are fully human, without the point mutations that raise concerns about immunogenicity.

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Figure 1: Intron and exon classes.
Figure 2: Domain structures and splice frame diagrams of the regulatory proteases of blood coagulation and fibrinolysis.
Figure 3: In vitro exon shuffling.

References

  1. 1

    Gilbert, W. Why genes in pieces? Nature 271, 501 (1978).

  2. 2

    Kuiper, J. et al. Interaction of mutants of tissue-type plasminogen activator with liver cells: effect of domain deletions. Biochem. J. 313, 775–780 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3

    Browne, M.J. et al. A tissue-type plasminogen activator mutant with prolonged clearance in vivo. Effect of removal of the growth factor domain. J. Biol. Chem. 263, 1599–1602 (1988).

    CAS  PubMed  Google Scholar 

  4. 4

    Doolittle, R.F. The multiplicity of domains in proteins. Annu. Rev. Biochem. 64, 287–314 (1995).

    CAS  PubMed  Article  Google Scholar 

  5. 5

    Bork, P. Mobile modules and motifs. Curr. Opin. Struct. Biol. 2, 413–421 (1992).

    CAS  Article  Google Scholar 

  6. 6

    Patthy, L. Modular exchange principles in proteins. Curr. Opin. Struct. Biol. 1, 351–361 (1991).

    CAS  Article  Google Scholar 

  7. 7

    Patthy, L. Introns and exons. Curr. Opin. Struct. Biol. 4, 383–392 (1994).

    CAS  Article  Google Scholar 

  8. 8

    Belfort, M. & Perlman, P.S. Mechanisms of intron mobility. J. Biol. Chem. 270, 30237–30240 (1995).

    CAS  PubMed  Article  Google Scholar 

  9. 9

    Patthy, L. Intron-dependent evolution: preferred types of exons and introns. FEBS Lett. 214, 1–7 (1987).

    CAS  PubMed  Article  Google Scholar 

  10. 10

    Sharp, P.A. Speculations on RNA splicing. Cell 23, 643–646 (1981).

    CAS  PubMed  Article  Google Scholar 

  11. 11

    Patthy, L. Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. Cell 41, 657–663 (1985).

    CAS  PubMed  Article  Google Scholar 

  12. 12

    Tordai, H., Banyai, L. & Patthy, L. The PAN module: the N-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins. FEBS Lett. 461, 63–67 (1999).

    CAS  PubMed  Article  Google Scholar 

  13. 13

    Gitschier, J. et al. Characterization of the human factor VIII gene. Nature 312, 326–330 (1984).

    CAS  PubMed  Article  Google Scholar 

  14. 14

    Cripe, L.D., Moore, K.D., & Kane, W.H. Structure of the gene for human coagulation factor V. Biochemistry 31, 3777–3385 (1992).

    CAS  PubMed  Article  Google Scholar 

  15. 15

    Bottenus, R.E., Ichinose, A. &, Davie, E.W. Nucleotide sequence of the gene for the b subunit of human factor XIII. Biochemistry 29, 11195–11209 (1990).

    CAS  PubMed  Article  Google Scholar 

  16. 16

    Mancuso, D.J. et al. Structure of the gene for human von Willebrand factor. J. Biol. Chem. 264, 19514–19527 (1989).

    CAS  PubMed  Google Scholar 

  17. 17

    Crameri, A., Cwirla, S. & Stemmer, W.P. Construction and evolution of antibody-phage libraries by DNA shuffling. Nat. Med. 2, 100–102 (1996).

    CAS  PubMed  Article  Google Scholar 

  18. 18

    Lollar, P., Parker, E.T. & Fay P.J. Coagulant properties of hybrid human/porcine factor VIII molecules. J. Biol. Chem. 267, 23652–23657 (1992).

    CAS  PubMed  Google Scholar 

  19. 19

    Chang, J.Y., Monroe, D.M., Stafford, D.W., Brinkhous, K.M. & Roberts, H.R. Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B. J. Clin. Invest. 100, 886–892 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20

    Langer-Safer, P.R. et al. Replacement of finger and growth factor domains of tissue plasminogen activator with plasminogen kringle 1. Biochemical and pharmacological characterization of a novel chimera containing a high affinity fibrin-binding domain linked to a heterologous protein. J. Biol. Chem. 266, 3715–3723 (1991).

    CAS  PubMed  Google Scholar 

  21. 21

    Bell, G.I. et al. Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization. Nucleic Acids Res. 14, 8427–8446 (1986).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22

    Ny, T., Elgh, F. & Lund, B. The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains. Proc. Natl. Acad. Sci. USA 81, 5355–5359 (1984).

    CAS  PubMed  Article  Google Scholar 

  23. 23

    Leytus, S.P., Foster, D.C., Kurachi, K. & Davie, E.W. Gene for human factor X: a blood coagulation factor whose gene organization is essentially identical with that of factor IX and protein C. Biochemistry 25, 5098–5102 (1986).

    CAS  PubMed  Article  Google Scholar 

  24. 24

    Miyazawa, K., Kitamura, A. & Kitamura, N. Structural organization and the transcription initiation site of the human hepatocyte growth factor gene. Biochemistry 30, 9170–9176 (1991).

    CAS  PubMed  Article  Google Scholar 

  25. 25

    Degen, S.J. & Davie, E.W. Nucleotide sequence of the gene for human prothrombin. Biochemistry 26, 6165–6177 (1987).

    CAS  PubMed  Article  Google Scholar 

  26. 26

    Patel, R.S., Odermatt, E., Schwarzbauer, J.E. & Hynes, R.O. Organization of the fibronectin gene provides evidence for exon shuffling during evolution. EMBO J. 6, 2565–2572 (1987).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27

    Cool, D.E. & MacGillivray, R.T. Characterization of the human blood coagulation factor XII gene. Intron/exon gene organization and analysis of the 5′-flanking region. J. Biol. Chem. 262, 13662–13673 (1987).

    CAS  PubMed  Google Scholar 

  28. 28

    Kim, S.J., Ruiz, N., Bezouska, K. & Drickamer K. Organization of the gene encoding the human macrophage mannose receptor (MRC1). Genomics 14, 721–727 (1992).

    CAS  PubMed  Article  Google Scholar 

  29. 29

    Schwarzbauer, J.E., Patel, R.S., Fonda, D. & Hynes, R.O. Multiple sites of alternative splicing of the rat fibronectin gene transcript. EMBO J. 6, 2573–2580 (1987).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30

    Giger, R.J. et al. The gene of chicken axonin–1. Complete structure and analysis of the promoter. Eur. J. Biochem. 227, 617–628 (1995).

    CAS  PubMed  Article  Google Scholar 

  31. 31

    del Castillo, I., Cohen-Salmon, M., Blanchard, S., Lutfalla, G. & Petit, C. Structure of the X-linked Kallmann syndrome gene and its homologous pseudogene on the Y chromosome. Nat. Genet. 2, 305–310 (1992).

    CAS  PubMed  Article  Google Scholar 

  32. 32

    Petersen, T.E., Martzen, M.R., Ichinose, A. & Davie, E.W. Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system. J. Biol. Chem. 265, 6104–6111 (1990).

    CAS  PubMed  Google Scholar 

  33. 33

    Beaubien, G. et al. Gene structure and chromosomal localization of plasma kallikrein. Biochemistry 30, 1628–1635 (1991).

    CAS  PubMed  Article  Google Scholar 

  34. 34

    Asakai, R., Davie, E.W. & Chung, D.W. Organization of the gene for human factor XI. Biochemistry 26 7221–7228 (1987).

    CAS  PubMed  Article  Google Scholar 

  35. 35

    Schmidel, D.K., Tatro, A.V., Phelps, L.G., Tomczak, J.A. & Long, G.L. Organization of the human protein S genes. Biochemistry 29, 7845–7852 (1990).

    CAS  PubMed  Article  Google Scholar 

  36. 36

    Edenbrandt, C.M., Lundwall, A., Wydro, R. & Stenflo, J. Molecular analysis of the gene for vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization. Biochemistry 29, 7861–7868 (1990).

    CAS  PubMed  Article  Google Scholar 

  37. 37

    Ploos van Amstel, H.K., Reitsma, P.H., van der Logt C.P. & Bertina, R.M. Intron-exon organization of the active human protein S gene PS alpha and its pseudogene PS beta: duplication and silencing during primate evolution. Biochemistry 29 7853–7861 (1990).

    CAS  PubMed  Article  Google Scholar 

  38. 38

    Johnston, G.I., Bliss, G.A., Newman, P.J. & McEver, R.P. Structure of the human gene encoding granule membrane protein–140, a member of the selectin family of adhesion receptors for leukocytes. J. Biol. Chem. 265, 21381–21385 (1990).

    CAS  PubMed  Google Scholar 

  39. 39

    Bensi, G., Raugei, G., Klefenz, H. & Cortese, R. Structure and expression of the human haptoglobin locus. EMBO J. 4, 119–126 (1985).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40

    Girard, T.J. et al. Structure of the human lipoprotein-associated coagulation inhibitor gene. Intro/exon gene organization and localization of the gene to chromosome 2. J. Biol. Chem. 266, 5036–5041 (1991).

    CAS  PubMed  Google Scholar 

  41. 41

    Christiano, A.M. et al. Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene. Genomics 21, 169–179 (1994).

    CAS  PubMed  Article  Google Scholar 

  42. 42

    Creighton, T.E. & Charles, I.G. Sequences of the genes and polypeptide precursors for two bovine protease inhibitors. J. Mol. Biol. 194, 11–22 (1987).

    CAS  PubMed  Article  Google Scholar 

  43. 43

    Shimasaki, S. et al. Primary structure of the human follistatin precursor and its genomic organization. Proc. Natl. Acad. Sci. USA 85, 4218–4222 (1988).

    CAS  PubMed  Article  Google Scholar 

  44. 44

    Villarreal, X.C., Mann, K.G. & Long, G.L. Structure of human osteonectin based upon analysis of cDNA and genomic sequences. Biochemistry 28, 6483–6491 (1989).

    CAS  PubMed  Article  Google Scholar 

  45. 45

    Scott, M.J. et al. Ovoinhibitor introns specify functional domains as in the related and linked ovomucoid gene. J. Biol. Chem. 262, 5899–5907 (1987).

    CAS  PubMed  Google Scholar 

  46. 46

    Gott, P. et al. Human trefoil peptides: genomic structure in 21q22.3 and coordinated expression. Eur. J. Hum. Genet. 4, 308–315 (1996).

    CAS  PubMed  Article  Google Scholar 

  47. 47

    Metsaranta, M., Toman, D., de Crombrugghe, B. & Vuorio, E. Mouse type II collagen gene. Complete nucleotide sequence, exon structure, and alternative splicing. J. Biol. Chem. 266, 16862–16869 (1991).

    CAS  PubMed  Google Scholar 

  48. 48

    Martinerie, C. et al. Structural analysis of the human nov proto-oncogene and expression in Wilms tumor. Oncogene 9, 2729–2732 (1994).

    CAS  PubMed  Google Scholar 

  49. 49

    Lawler, J. et al. Characterization of the murine thrombospondin gene. Genomics 11, 587–600 (1991).

    CAS  PubMed  Article  Google Scholar 

  50. 50

    Ogata, R.T., Rosa, P.A. & Zepf, N.E. Sequence of the gene for murine complement component C4. J. Biol. Chem. 264, 16565–16572 (1989).

    CAS  PubMed  Google Scholar 

  51. 51

    Grassel, S., Sicot, F.X., Gotta, S. & Chu, M.L. Mouse fibulin-2 gene. Complete exon-intron organization and promoter characterization. Eur. J. Biochem. 263, 471–477 (1999).

    CAS  PubMed  Article  Google Scholar 

  52. 52

    Takahara, K., Lee, S., Wood, S. & Greenspan, D.S. Structural organization and genetic localization of the human bone morphogenetic protein 1/mammalian tolloid gene. Genomics 29, 9–15 (1995).

    CAS  PubMed  Article  Google Scholar 

  53. 53

    Rossignol, M., Gagnon, M.L. & Klagsbrun, M. Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms. Genomics 70, 211–222 (2000).

    CAS  PubMed  Article  Google Scholar 

  54. 54

    Zimmermann, K., Hoischen, S., Hafner, M. & Nischt, R. Genomic sequences and structural organization of the human nidogen gene (NID). Genomics 27, 245–250 (1995).

    CAS  PubMed  Article  Google Scholar 

  55. 55

    Parma, J., Christophe, D., Pohl, V. & Vassart, G. Structural organization of the 5′ region of the thyroglobulin gene. Evidence for intron loss and “exonization” during evolution. J. Mol. Biol. 196, 769–779 (1987).

    CAS  PubMed  Article  Google Scholar 

  56. 56

    Linnenbach, A.J. et al. Retroposition in a family of carcinoma-associated antigen genes. Mol. Cell. Biol. 13, 1507–1515 (1993).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57

    Sudhof, T.C., Goldstein, J.L., Brown, M.S. & Russell, D.W. The LDL receptor gene: a mosaic of exons shared with different proteins. Science 228, 815–822 (1985).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. 58

    Marazziti, D., Eggertsen, G., Fey, G.H. & Stanley, K.K. Relationships between the gene and protein structure in human complement component C9. Biochemistry 27, 6529–6534 (1988).

    CAS  PubMed  Article  Google Scholar 

  59. 59

    Kirschbaum, N.E., Gumina, R.J. & Newman, P.J. Organization of the gene for human platelet/endothelial cell adhesion molecule-1 shows alternatively spliced isoforms and a functionally complex cytoplasmic domain. Blood 84, 4028–4037 (1994).

    CAS  PubMed  Google Scholar 

  60. 60

    Coutelle, O. et al. The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu. Gene 208, 7–15 (1998).

    CAS  PubMed  Article  Google Scholar 

  61. 61

    Nolan, K.F., Kaluz, S., Higgins, J.M., Goundis, D. & Reid, K.B. Characterization of the human properdin gene. Biochem. J. 287, 291–297 (1992).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. 62

    Kiss, I. et al. Structure of the chicken link protein gene: exons correlate with the protein domains. Proc. Natl. Acad. Sci. USA 84, 6399–6403 (1987).

    CAS  PubMed  Article  Google Scholar 

  63. 63

    Screaton, G.R. et al. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. USA 89, 12160–12164 (1992).

    CAS  PubMed  Article  Google Scholar 

  64. 64

    Naso, M.F., Zimmermann, D.R. & Iozzo, R.V. Characterization of the complete genomic structure of the human versican gene and functional analysis of its promoter. J. Biol. Chem. 269, 32999–33008 (1994).

    CAS  PubMed  Google Scholar 

  65. 65

    Ord, D.C. et al. Structure of the gene encoding the human leukocyte adhesion molecule–1 (TQ1, Leu-8) of lymphocytes and neutrophils. J. Biol.Chem. 265, 7760–7767 (1990).

    CAS  PubMed  Google Scholar 

  66. 66

    Collins, T. et al. Structure and chromosomal location of the gene for endothelial-leukocyte adhesion molecule 1. J. Biol. Chem. 266, 2466–2473 (1991).

    CAS  PubMed  Google Scholar 

  67. 67

    Hoyle, G.W. & Hill, R.L. Structure of the gene for a carbohydrate-binding receptor unique to rat Kupffer cells. J. Biol. Chem. 266, 1850–1857 (1991).

    CAS  PubMed  Google Scholar 

  68. 68

    Hahn, D., Illisson, R., Metspalu, A. & Sterchi, E.E. Human N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase (human meprin): genomic structure of the alpha and beta subunits. Biochem. J. 346, 83–91 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. 69

    Kiss, I. et al. Structure of the gene for cartilage matrix protein, a modular protein of the extracellular matrix. Exon/intron organization, unusual splice sites, and relation to alpha chains of beta 2 integrins, von Willebrand factor, complement factors B and C2, and epidermal growth factor. J. Biol. Chem. 264, 8126–8134 (1989).

    CAS  PubMed  Google Scholar 

  70. 70

    Hayman, A.R., Koppel, J. & Trueb, B. Complete structure of the chicken alpha 2(VI) collagen gene. Eur. J. Biochem. 197, 177–184 (1991).

    CAS  PubMed  Article  Google Scholar 

  71. 71

    Corbi A.L., Garcia–Aguilar, J. & Springer, T.A. Genomic structure of an integrin alpha subunit, the leukocyte p150,95 molecule. J. Biol. Chem. 265, 2782–2788 (1990).

    CAS  PubMed  Google Scholar 

  72. 72

    Jenne, D. & Stanley, K.K. Nucleotide sequence and organization of the human S-protein gene: repeating peptide motifs in the "pexin" family and a model for their evolution. Biochemistry 26, 6735–6742 (1987).

    CAS  PubMed  Article  Google Scholar 

  73. 73

    Campbell, S.M., Rosen, J.M., Hennighausen, L.G., Strech-Jurk, U. & Sippel, A.E. Comparison of the whey acidic protein genes of the rat and mouse. Nucleic Acids Res. 12, 8685–8697 (1984).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. 74

    Stetler, G., Brewer, M.T. & Thompson, R.C. Isolation and sequence of a human gene encoding a potent inhibitor of leukocyte proteases. Nucleic Acids Res. 14, 7883–7896 (1986).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  75. 75

    Altruda, F., Poli, V., Restagno, G. & Silengo, L. Structure of the human hemopexin gene and evidence for intron-mediated evolution. J. Mol. Evol. 27, 102–108 (1988).

    CAS  PubMed  Article  Google Scholar 

  76. 76

    Huhtala, P., Chow, L.T. & Tryggvason, K. Structure of the human type IV collagenase gene. J. Biol. Chem. 265, 11077–11082 (1990).

    CAS  PubMed  Google Scholar 

  77. 77

    Brauninger, A., Karn, T., Strebhardt, K. & Rubsamen-Waigmann, H. Characterization of the human CSK locus. Oncogene 8, 1365–1369 (1993).

    CAS  PubMed  Google Scholar 

  78. 78

    Rohrer, J., Parolini, O., Belmont, J.W. & Conley, M.E. The genomic structure of human BTK, the defective gene in X-linked agammaglobulinemia. Immunogenetics 40, 319–324 (1994).

    CAS  PubMed  Article  Google Scholar 

  79. 79

    Tanaka, A. et al. DNA sequence encoding the amino-terminal region of the human c-src protein: implications of sequence divergence among src-type kinase oncogenes. Mol. Cell. Biol. 7, 1978–1983 (1987).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  80. 80

    Anderson, S.K. et al. Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src. Mol. Cell. Biol. 5, 1122–1129 (1985).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  81. 81

    Airenne, T. et al. Structure of the human laminin gamma 2 chain gene (LAMC2): alternative splicing with different tissue distribution of two transcripts. Genomics 32, 54–64 (1996).

    CAS  PubMed  Article  Google Scholar 

  82. 82

    Kallunki, T., Ikonen, J., Chow, L.T., Kallunki, P. & Tryggvason, K. Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene. J. Biol. Chem. 266, 221–228 (1991).

    CAS  PubMed  Google Scholar 

  83. 83

    Vuolteenaho, R., Chow, L.T. & Tryggvason, K. Structure of the human laminin B1 chain gene. J. Biol. Chem. 265, 15611–15616 (1990).

    CAS  PubMed  Google Scholar 

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Correspondence to Willem P.C. Stemmer.

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Kolkman, J., Stemmer, W. Directed evolution of proteins by exon shuffling. Nat Biotechnol 19, 423–428 (2001). https://doi.org/10.1038/88084

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