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Libraries of hybrid proteins from distantly related sequences

Nature Biotechnologyvolume 19pages456460 (2001) | Download Citation



We introduce a method for sequence homology–independent protein recombination (SHIPREC) that can create libraries of single-crossover hybrids of unrelated or distantly related proteins. The method maintains the proper sequence alignment between the parents and introduces crossovers mainly at structurally related sites distributed over the aligned sequences. We used SHIPREC to create a library of interspecies hybrids of a membrane-associated human cytochrome P450 (1A2) and the heme domain of a soluble bacterial P450 (BM3). By fusing the hybrid gene library to the gene for chloramphenicol acetyl transferase (CAT), we were able to select for soluble and properly folded protein variants. Screening for 1A2 activity (deethylation of 7-ethoxyresorufin) identified two functional P450 hybrids that were more soluble in the bacterial cytoplasm than the wild-type 1A2 enzyme.

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  1. 1

    Stemmer, W.P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).

  2. 2

    Zhao, H., Giver, L., Shao, Z., Affholter, J.A. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261 (1998).

  3. 3

    Shao, Z., Zhao, H., Giver, L. & Arnold, F.H. Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic Acids Res. 26, 681–683 (1998).

  4. 4

    Volkov, A.A., Shao, Z. & Arnold, F.H. Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair. Nucleic Acids Res. 27, e18 (1999).

  5. 5

    Cherry, J.R. et al. Directed evolution of a fungal peroxidase. Nat. Biotechnol. 17, 379–384 (1999).

  6. 6

    Ostermeier, M., Shim, J.H. & Benkovic, S.J. A combinatorial approach to hybrid enzymes independent of DNA homology. Nat. Biotechnol. 17, 1205–1209 (1999).

  7. 7

    Mitra, B. et al. A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida. Biochemistry 32, 12959–12967 (1993).

  8. 8

    Shimoji, M., Yin, H., Higgins, L. & Jones, J.P. Design of a novel P450: a functional bacterial–human cytochrome P450 chimera. Biochemistry 37, 8848–8852 (1998).

  9. 9

    Bogarad, L.D. & Deem, M.W. A hierarchical approach to protein molecular evolution. Proc. Natl. Acad. Sci. USA 96, 2591–2595 (1999).

  10. 10

    Quattrochi, L.C., Okino, S.T., Pendurthi, U.R. & Tukey, R.H. Cloning and isolation of human cytochrome P-450 cDNAs homologous to dioxin-inducible rabbit mRNAs encoding P-450-4 and P-450-6. DNA 4, 395–400 (1985).

  11. 11

    Narhi, L.O., Kim, B.H., Stevenson, P.M. & Fulco, A.J. Partial characterization of a barbiturate-induced cytochrome P-450- dependent fatty acid monooxygenase from Bacillus megaterium. Biochem. Biophys. Res. Commun. 116, 851–858 (1983).

  12. 12

    Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989).

  13. 13

    Graf, R. & Schachman, H.K. Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase. Proc. Natl. Acad. Sci. USA 93, 11591–11596 (1996).

  14. 14

    Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescence proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

  15. 15

    Maxwell, K.L., Mittermaier, A.K., Forman-Kay, J.D. & Davidson, A.R. A simple in vivo assay for increased protein solubility. Protein Sci. 8, 1908–1911 (1999).

  16. 16

    Shaw, W.V. & Leslie, A.G. Chloramphenicol acetyltransferase. Ann. Rev. Biophys. Chem. 20, 363–386 (1991).

  17. 17

    Schenkman, J.B. & Janson, I. Spectral analysis of cytochromes P450. In Cytochrome P450 protocols, Vol. 107. (eds Phillips, I.R. & Shephard, E.A.) 25–33 (Humana Press Inc., Totowa, N.J.; 1998).

  18. 18

    Chang, T.K.H. & Waxman, D.J. Catalytic assays for human cytochrome P450. In Cytochrome P450 protocols, Vol. 107. (eds Phillips, I.R. & Shephard, E.A.) 85–93 (Humana Press Inc., Totowa, N.J; 1998).

  19. 19

    Dong, M.S., Yamazaki, H., Guo, Z. & Guengerich, F.P. Recombinant human cytochrome P450 1A2 and an N-terminal-truncated form: construction, purification, aggregation properties, and interactions with flavodoxin, ferredoxin, and NADPH-cytochrome P450 reductase. Arch. Biochem. Biophys. 327, 11–19 (1996).

  20. 20

    Bellamine, A., Gautier, J.C., Urban, P. & Pompon, D. Chimeras of the human cytochrome P450 1A family produced in yeast. Accumulation in microsomal membranes, enzyme kinetics and stability. Eur. J. Biochem. 225, 1005–1013 (1994).

  21. 21

    Ostermeier, M. & Benkovic, S.J. Evolution of protein function by domain swapping. Adv. Protein Chem. 55, 29–77 (2000).

  22. 22

    Parikh, A., Gillam, E.M. & Guengerich, F.P. Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784–788 (1997).

  23. 23

    Woodard, S.I. & Dailey, H.A. Regulation of heme biosynthesis in Escherichia coli. Arch. Biochem. Biophys. 316, 110–115 (1995).

  24. 24

    Jenkins, C.M., Pikuleva, I. & Waterman, M.R. Expression of eukaryotic cytochromes P450 in E. coli. In Cytochrome P450 protocols, Vol. 107. (eds Phillips, I.R. & Shephard, E.A.) 181–193 (Humana Press Inc., Totowa, N.J.; 1998).

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V.S. was supported by a research fellowship from the Deutsche Forschungsgemeinschaft (DGF). This work was funded by Maxygen Inc.

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  1. Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA, 91105

    • Volker Sieber
    • , Carlos A. Martinez
    •  & Frances H. Arnold


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Correspondence to Frances H. Arnold.

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