Research Paper | Published:

A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification

Bio/Technologyvolume 6pages12041210 (1988) | Download Citation



A small hydrophilic peptide of eight amino acids (AspTyrLysAspAspAspAspLys) was engineered onto the N-terminus of a variety of recombinant lymphokines for the purpose of aiding in their detection and purification from yeast supernatants or E. coli extracts. An antibody specific for the first four amino acids of this sequence was used as a detection reagent and for immunoaffinity purification of products under mild conditions. Because of the small size of the peptide moiety and its hydrophilic nature, the proteins were unaffected by its presence and retained a high level of biological activity. In addition, it was possible to remove the peptide via an enzymatic cleavage procedure using enterokinase.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    Itakura, K., Hirose, T., Crea, R., Riggs, A.D., Heyneker, H.L., Bolivar, F., and Boyer, H.W. 1977. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198:1056–1063.

  2. 2

    Goeddel, D.V., Kleid, D.G., Bolivar, F., Heyneker, H.L., Yansura, D.G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K., and Riggs, A.D. 1979. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc. Natl. Acad. Sci. USA 76:106–110.

  3. 3

    Brake, A.J., Merryweather, J.P., Coit, D.G., Heberlein, U.A., Masiarz, F.R., Mullenback, G.T., Urdea, M.S., Valenzuela, P., and Barr, P.J. 1984. α-Factor-directed synthesis and secretion of foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81:4642–4646.

  4. 4

    Villa-Komaroff, L., Efstratiadis, A., Broome, S., Lomedico, P., Tizard, R., Naber, S.P., Chick, W.L., and Gilbert, W. 1978. A bacterial clone synthesizing proinsulin. Proc. Natl. Acad. Sci. USA 75:3727–3731.

  5. 5

    Germino, J. and Bastia, D. 1984. Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis. Proc. Natl. Acad. Sci. USA 81:4692–4696.

  6. 6

    Abrahmsen, L., Moks, T., Nilsson, B., and Uhlen, M. 1986. Secretion of heterologous gene products to the culture medium of Escherichia coli. Nucleic Acids Res. 14:7487–7500.

  7. 7

    Munro, S. and Pelham, H.R.B. 1984. Use of peptide tagging to detect proteins expressed from cloned genes. EMBO J. 3:3087–3093.

  8. 8

    Ikehara, M., Ohtsuka, E., Tokunaga, T., Nishikawa, S., Uesugi, S., Tanaka, T., Aoyama, Y., Kikyodani, S., Fujimoto, K., Yanase, K., Fuchimura, K., and Morioka, H. 1986. Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences. Biochemistry 83:4695–4699.

  9. 9

    Wilchek, M., Miron, T., and Kohn, J. 1984. Affinity chromatography. Methods in Enzymol. 104::3–55.

  10. 10

    Nagai, K., and Thogerson, H.C. 1987. Synthesis and sequence specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 153:461–481.

  11. 11

    Schoner, R.G., Ellis, L.F., and Schoner, B.E. 1985. Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/Technology 3:151–154.

  12. 12

    Sung, W.L., Yao, F.L., Zahab, D.M., and Narang, S.A. 1986. Short synthetic oligodeoxyribonucleotide leader sequences enhance accumulation of human proinsulin synthesized in Escherichia coli. Proc. Natl. Acad. Sci. USA 83:561–565.

  13. 13

    Nilsson, B., Holmgren, E., Josephson, S., Gatenbeck, S., Philipson, L., and Uhlen, M. 1985. Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci. Nucleic Acids Res. 13:1151–1162.

  14. 14

    Moks, T., Abrahmsen, L., Osterlof, B., Josephson, S., Ostling, M., Enfors, S., Persson, I., Nilsson, B., and Uhlen, M. 1987. Large-scale affinity purification of human insulin-like growth factor I from culture medium of Escherichia coli. Bio/Technology 5:379–382.

  15. 15

    Sassenfeld, H.M. and Brewer, S.J. 1984. A polypeptide fusion designed for the purification of recombinant proteins. Bio/Technology 2:76–81.

  16. 16

    Maroux, S., Baratti, J., and Desnuelle, P. 1971. Purification and specificity of procine enterokinase. J. Biol. Chem. 246:5031–5039.

  17. 17

    Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N., Yoshimoto, R., and Hamuro, J. 1983. Structure and expression of a cloned cDNA for human interleukin-2. Nature 302:305–310.

  18. 18

    Nagata, S., Tsuchiya, M., Asano, S., Kaziro, Y., Yamazaki, T., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., and Ono, M. 1986. Molecular cloning and expression of cDNA for human granulocyte colony stimulating factor. Nature 319:415–417.

  19. 19

    Yang, Y-C., Ciarletta, A.B., Temple, P.A., Chung, M.P., Kovacic, S., Witek-Giannotti, J.S., Leary, A.C., Kriz, R., Donahue, R.E., Wong, G.G., and Clark, S.C. 1986. Human IL-3 (multi-CSF): Identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 47:3–10.

  20. 20

    Yokota, T., Otsuka, T., Mosmann, T., Banchereau, J., DeFrance, T., Blanchard, D., DeVries, J.E., Lee, F., and Arai, K-I. 1986. Isolation and characterization of a human cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell and T-cell stimulating activities. Proc. Natl. Acad. Sci. USA 83:5894–5898.

  21. 21

    Cantrell, M.A., Anderson, D., Cerretti, D.P., Price, V., McKereghan, K., Tushinski, R.J., Mochizuki, D.Y., Larsen, A., Grabstein, K., Gillis, S., and Cosman, D. 1985. Cloning, sequence, and expression of a human granulocyte/macrophage colony stimulating factor. Proc. Natl. Acad. Sci. USA 82:6250–6254.

  22. 22

    Urdal, D.L., Mochizuki, D., Conlon, P.J., March, C.J., Remerowski, M.L., Eisenman, J., Ramthun, C., and Gillis, S. 1984. Lymphokine purification by reversed-phase high performance liquid chromatography. J. Chromatography 296;171–179.

  23. 23

    Maurer, P.H., Clark, L.G., and Liberti, P.A. 1970. Antigenicity of polypeptides (polyαamino acids): Calcium-dependent and independent antibodies. J. Immunol. 105:567–573.

  24. 24

    Hitzeman, R.A., Leung, D.W., Perry, L.J., Kohr, W.J., Levine, H.L., and Goeddel, D.V. 1983. Secretion of human interferons by yeast. Science 219:620–625.

  25. 25

    Aston, R., Cooper, L., Holder, A., Ivanyi, J., and Preece, M. 1985. Monoclonal antibodies to human growth hormone can distinguish between pituitary and genetically engineered forms. Molec. Immunol. 22:271–275.

  26. 26

    Kronheim, S.R., Cantrell, M.A., Deeley, M.C., March, C.J., Glackin, P.J., Anderson, D.M., Hemenway, T., Merriam, J.E., Cosman, D., and Hopp, T.P. 1986. Purification and characterization of human interleukin-1 expressed in Escherichia coli. Bio/Technology 4:1078–1082.

  27. 27

    Hopp, T.P. and Woods, K.R. 1981. Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78:3824–3828.

  28. 28

    Hopp, T.P. 1986. Protein surface analysis: Methods for identifying antigenic determinants and other interaction sites. J. Immunol. Methods 88::1–18.

  29. 29

    Sela, M. and Arnon, R. 1960. Studies on the chemical basis of the antigenicity of proteins. Biochem. J. 75:91–102.

  30. 30

    Liepnieks, J.J. and Light, A. 1979. The preparation and properties of bovine enterokinase. J. Biol. Chem. 254:1677–1683.

  31. 31

    Price, V., Mochizuki, D., March, C.J., Cosman, D., Deeley, M.C., Klinke, R., Clevenger, W., Gillis, S., Baker, P., and Urdal, D. 1987. Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine inter-leukin-2 from yeast. Gene 55:287–293.

  32. 32

    Beier, D.R. and Young, E.T. 1982. Characterization of a regulatory region upstream of the ADR2 locus of S. cerevisiae. Nature 300:724–728.

  33. 33

    Hinnen, A., Hicks, J.B., and Fink, G.R. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. USA 75:1929–1933.

  34. 34

    Libby, R.T., Braedt, G., Kronheim, S.R., March, C.J., Urdal, D.L., Chiaverotti, T.A., Tushinski, R.J., Mochizuki, D.Y., Hopp, T.P., and Cosman, D. 1987. Expression and purification of native human granulocyte-macrophage colony stimulating factor from an Escherichia coli vector. DNA 6:221–229.

  35. 35

    Hopp, T.P. 1984. Immunogenicity of a synthetic HBsAg peptide: Enhancement by conjugation to a fatty acid carrier. Molec. Immunol. 21:13–16.

  36. 36

    Gillis, S., Ferm, M.M., Ou, W., and Smith, K. 1978. T cell growth factor: parameters of production and a quantitative microassay for activity. J. Immunol. 120:2027–2033.

  37. 37

    Mochizuki, D.Y., Eisenman, J., Conlon, P.J., Park, L.S., and Urdal, D.L. 1986. Development and characterization of antiserum to murine granulocyte-macrophage colony stimulating factor. J. Immunol. 136:3760–3769.

  38. 38

    Prestidge, R.L., Watson, J.D., Urdal, D.L., Mochizuki, D., Conlon, P., and Gillis, S. 1984. Biochemical comparison of murine colony stimulating factors secreted by a T cell lymphoma and a myelomonocytic leukemia. J. Immunol. 133:293–298.

  39. 39

    Grabstein, K., Eisenman, J., Mochizuki, D., Shanebeck, K., Conlon, P., Hopp, T., March, C., and Gillis, S. 1986. Purification to homogeneity of B-cell stimulating factor: A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines. J. Exp. Med. 163:1405–1414.

  40. 40

    Metcalf, D. 1986. The molecular biology and functions of granulocyte-macrophage colony stimulating factors. Blood 67:257–267.

Download references

Author information

Author notes

    • Randell T. Libby

    Present address: Departments of Genetics, SK-50, and Pathology, SM-30, University of Washington, Seattle, WA, 98195


  1. Immunex Corporation, 51 University Street, Seattle, Washington, 98101

    • Thomas P. Hopp
    • , Kathryn S. Prickett
    • , Virginia L. Price
    • , Carl J. March
    • , Douglas Pat Cerretti
    • , David L. Urdal
    •  & Paul J. Conlon


  1. Search for Thomas P. Hopp in:

  2. Search for Kathryn S. Prickett in:

  3. Search for Virginia L. Price in:

  4. Search for Randell T. Libby in:

  5. Search for Carl J. March in:

  6. Search for Douglas Pat Cerretti in:

  7. Search for David L. Urdal in:

  8. Search for Paul J. Conlon in:

About this article

Publication history



Issue Date


Further reading