Molecular Diversity

, Volume 1, Issue 1, pp 29–38 | Cite as

Libraries of random-sequence polypeptides produced with high yield as carboxy-terminal fusions with ubiquitin

  • Thomas H. LaBean
  • Stuart A. Kauffman
  • Tauseef R. Butt
Research Papers


Libraries of random-sequence polypeptides have been shown to be valuable sources of novel molecules possessing a variety of useful biologic-like activities, some of which may hold promise as potential vaccines and therapeutics. Previous random peptide expression systems were limited to low levels of peptide production and often to short sequences. Here we describe a series of libraries designed for increased polypeptide length. Cloned as carboxy-terminal extensions of ubiquitin, the fusions were produced inE. coli at high levels, and were purified to homogeneity. The majority of the extension proteins examined could be cleaved from ubiquitin by treatment with a ubiquitin-fusion hydrolase. The libraries described here are appropriate sources of novel polypeptides with desired binding or catalytic function, as well as tools with which to examine inherent properties of proteins as a whole. Toward the latter goal, we have examined structural properties of random-sequence proteins purified from these libraries. Quite surprisingly, fluorescence emission spectra of intrinsic tryptophan residues in several purified fusion proteins, under native-like and denaturing conditions, often resemble those expected for folded and unfolded states, respectively. The results presented here detail an important expansion in the range of potential uses for random-sequence polypeptide libraries.


Combinatorial library Random-sequence polypeptide Ubiquitin Ubiquitin fusion Protein folding 


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  1. 1.
    Scott, J.K.,Discovering peptide ligands using epitope libraries, Trends Biochem. Sci., 17 (1992) 241–245.Google Scholar
  2. 2.
    Kauffman, S.A.,Applied molecular evolution, J. Theor. Biol., 157 (1992) 1–7.Google Scholar
  3. 3.
    Wetzel, R.,Learning from the immune system: Laboratory methods for creating and refining molecular diversity in polypeptides, Protein Eng., 4 (1991) 371–374.Google Scholar
  4. 4.
    Geysen, H.M., Rodda, S.J. and Mason, T.J.,A priori delineation of a peptide which mimics a discontinuous antigenic determinant, Mol. Immunol., 23 (1986) 709–715.Google Scholar
  5. 5.
    Houghten, R.A., Pinilla, C., Blondelle, S.E., Appel, J.R., Dooley, C.T. and Cuervo, J.H.,Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery, Nature, 354 (1991) 84–86.Google Scholar
  6. 6.
    Mandecki, W.,A method for construction of long randomized open reading frames and polypeptides, Protein Eng., 3 (1990) 221–226.Google Scholar
  7. 7.
    Cull, M.G., Miller, J.F. and Schatz, P.J.,Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor, Proc. Natl. Acad. Sci. USA, 89 (1992) 1865–1869.Google Scholar
  8. 8.
    Scott, J.K. and Smith, G.P.,Searching for peptide ligands with an epitope library, Science, 249 (1990) 386–390.Google Scholar
  9. 9.
    Cwirla, S.E., Peters, E.A., Barrett, R.W. and Dower, W.J.,Peptides on phage: A vast library of peptides for identifying ligands, Proc. Natl. Acad. Sci. USA, 87 (1990) 6378–6382.Google Scholar
  10. 10.
    Devlin, J.J., Panganiban, L.C. and Devlin, P.E.,Random peptide libraries: A source of specific protein binding molecules, Science, 249 (1990) 404–406.Google Scholar
  11. 11.
    McCafferty, J., Jackson, R.H. and Chiswell, D.J.,Phage-enzymes: Expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage, Protein Eng., 4 (1991) 955–961.Google Scholar
  12. 12.
    Corey, D.R., Shiaw, A.K., Yang, Q., Janowski, B.A. and Craik, C.S.,Trypsin display on the surface of bacteriophage, Gene, 128 (1993) 129–134.Google Scholar
  13. 13.
    Makowski, L.,Structural constraints on the display of foreign peptides on filamentous bacteriophages, Gene, 128 (1993) 5–11.Google Scholar
  14. 14.
    Butt, T.R., Jonnalagadda, S., Monia, B.P., Sternberg, E.J., Marsh, J.A., Stadel, I.M., Ecker, D.J. and Crooke, S.T.,Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli, Proc. Natl. Acad. Sci. USA, 86 (1989) 2540–2544.Google Scholar
  15. 15.
    Yoo, Y., Rote, K. and Rechsteiner, M.,Synthesis of peptides as cloned ubiquitin extensions, J. Biol. Chem., 264 (1989) 17078–17083.Google Scholar
  16. 16.
    Wittliff, J., Wenz, L., Dong, J., Nawaz, Z. and Butt, T.R.,Expression and characterization of an active human estrogen receptor as a ubiquitin fusion from E. coli, J. Biol. Chem., 265 (1990) 22016–22022.Google Scholar
  17. 17.
    Hershko, A. and Ciechanover, A.,The ubiquitin system for protein degradation, Annu. Rev. Biochem., 61 (1992) 761–807.Google Scholar
  18. 18.
    Monia, B.P., Ecker, D.J. and Crooke, S.T.,New perspectives on the structure and function of ubiquitin, Biotechnology, 8 (1990) 209–215.Google Scholar
  19. 19.
    Wolf, S., Lottspeich, F. and Baumeister, W.,Ubiquitin found in the archaebacterium Thermoplasma acidophilum, FEBS Lett., 326 (1993) 42–44.Google Scholar
  20. 20.
    Finley, D., Bartel, B. and Varshavsky, A.,The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis, Nature, 338 (1989) 394–401.Google Scholar
  21. 21.
    Redman, K.J. and Rechsteiner, M.,Identification of the long ubiquitin extension as ribosomal protein S27a, Nature, 338 (1989) 438–440.Google Scholar
  22. 22.
    Ecker, D.J., Stadel, J.M., Butt, T.R., Marsh, J.A., Monia, B.P., Powers, D.A., Gorman, J.A., Clark, P.E., Warren, F., Shatzman, A. and Crooke, S.T.,Increasing gene expression in yeast by fusion to ubiquitin, J. Biol. Chem., 264 (1989) 7715–7719.Google Scholar
  23. 23.
    Vijay-Kumar, S., Bugg, C.E. and Cook, W.J.,Structure of ubiquitin refined at 1.8 Å resolution, J. Mol. Biol., 194 (1987) 531–544.Google Scholar
  24. 24.
    DiStefano, D.L. and Wand, A.J.,Two-dimensional 1H NMR study of human ubiquitin: A main chain directed assignment and structure analysis, Biochemistry, 26 (1987) 7272–7281.Google Scholar
  25. 25.
    Cary, P.D., King, D.S., Crane-Robinson, C., Bradbury, E.M., Rabbani, A., Goodwin, G.H. and Johns, E.W.,Structural studies on two high-mobility-group proteins from calf thymus, HMG-14 and HMG-20 (ubiquitin), and their interaction with DNA, Eur. J. Biochem., 112 (1980) 577–580.Google Scholar
  26. 26.
    Lenkinski, R.E., Chen, D.M., Glickson, J.D. and Goldstein, G.,Nuclear magnetic resonance studies of the denaturation of ubiquitin, Biochim. Biophys. Acta, 494 (1977) 126–130.Google Scholar
  27. 27.
    Wilkinson, K.D., Lee, K.M., Deshpande, S., Duerksen-Hughes, P., Boss, J.M. and Pohl, J.,The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase, Science, 246 (1989) 670–673.Google Scholar
  28. 28.
    Baker, R.T., Tobias, J.W. and Varshavsky, A.,Ubiquitin-specific proteases of S. cerevisiae: Cloning of UBP2 and UBP3, and analysis of the UBP gene family, J. Biol. Chem., 267 (1992) 23364–23375.Google Scholar
  29. 29.
    LaBean, T.H. and Kauffman, S.A.,Design of synthetic gene libraries encoding random sequence proteins with desired ensemble characteristics, Protein Sci., 2 (1993) 1249–1254.Google Scholar
  30. 30.
    LaBean, T.H., Kauffman, S.A. and Butt, T.R.,Evidence of folded structure in random-sequence polypeptides, Nature Struct. Biol., manuscript submitted for publication.Google Scholar
  31. 31.
    Kamtekar, S., Schiffer, J., Xiong, H., Babik, J. and Hecht, M.H.,Protein design by binary patterning of polar and nonpolar amino acids, Science, 262 (1993) 1680–1685.Google Scholar
  32. 32.
    Davidson, A.R. and Sauer, R.T.,Folded proteins occur frequently in libraries of random amino acid sequences, Proc. Natl. Acad. Sci. USA, 91 (1994) 2146–2150.Google Scholar
  33. 33.
    Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.Google Scholar
  34. 34.
    Ecker, D.J., Butt, T.R., Marsh, J.A., Sternberg, E.J., Margolis, N., Monia, B.P., Jonnalagadda, S., Khan, M.I., Weber, P.L., Mueller, L. and Crooke, S.T.,Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin, J. Biol. Chem., 262 (1987) 14213–14221.Google Scholar
  35. 35.
    Meselson, M. and Yuan, R.,DNA restriction enzyme from E. coli, Nature, 217 (1968) 1110–1114.Google Scholar
  36. 36.
    Mott, J.E., Grant, R.A., Ho, Y.S. and Platt, T.,Maximizing gene expression from plasmid vectors containing the lambda PL promoter: Strategies for overproducing transcription termination factor rho, Proc. Natl. Acad. Sci. USA, 82 (1985) 88–92.Google Scholar
  37. 37.
    Shatzman, A.R. and Rosenberg, M.,Efficient expression of heterologous genes in Escherichia coli. The pAS vector system and its applications, Ann. New York Acad. Sci., 478 (1986) 233–248.Google Scholar
  38. 38.
    Wilkinson, K.D., Cox, M.J., Mayer, A.N. and Frey, T.,Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase, Biochemistry, 25 (1986) 6644–6649.Google Scholar
  39. 39.
    DeGraaf, M.E., Miceli, R.M., Mott, J.E. and Fischer, H.D.,Biochemical diversity in aphage display library of random decapeptides, Gene, 128 (1993) 13–17.Google Scholar
  40. 40.
    Lakowicz, J.R., Principles of Fluorescence Spectroscopy, Plenum Press, New York, NY, 1983.Google Scholar
  41. 41.
    Butt, T.R. and Stadel, J.M.,Increasing expression of genes in heterologous systems by fusion with ubiquitin: The role of ubiquitin structure and folding, Protein Sci., manuscript submitted for publication.Google Scholar
  42. 42.
    Pearce, S.F. and Hawrot, E.,Intrinsic fluorescence fragments of nicotinic acetylcholine receptor Perturbations produced upon binding alpha-bugarotoxin, Biochemistry, 29 (1990) 10649–10659.Google Scholar

Copyright information

© ESCOM Science Publishers B.V. 1995

Authors and Affiliations

  • Thomas H. LaBean
    • 1
  • Stuart A. Kauffman
    • 1
    • 2
  • Tauseef R. Butt
    • 1
    • 3
  1. 1.Department of Biochemistry and BiophysicsUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Santa Fe InstituteSanta FeUSA
  3. 3.Department of Molecular Virology and Host Defense, Research and DevelopmentSmithKline Beecham PharmaceuticalsKing of PrussiaUSA

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