Research Paper | Published:

Purification of Calf Prochymosin (Prorennin) Synthesized in Escherichia Coli

Bio/Technologyvolume 2pages800804 (1984) | Download Citation



Recombinant calf prochymosin synthesized in E. coli was shown to accumulate in the form of insoluble inclusion bodies. Isolation of this aggregated material, combined with specific washing procedures, was the most significant stage of the purification protocol. Disruption of proteins in the inclusion bodies necessitated denaturation and renaturation. The method described can completely solubilize prochymosin. At this stage acidification produced active chymosin. Subsequently, ion-exchange chromatography produced highly purified prochymosin which, after acidification, yielded chymosin >99% pure.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    Cheeseman, G.E. 1981. Rennet and Cheesemaking. p. 195–211. In: Enzymes and Food Processing. Birch, G. G., Blakeborough, N. and Parker, K.J. (eds). Applied Science Publishers Ltd., Essex, U.K.

  2. 2

    Harris, T.J.R., Lowe, P.A., Thomas, P.G., Eaton, M.A.W., Millican, T.A., Patel, T.P., Bose, C.C., Carey, N.H. and Doel, M.T. 1982. Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin. Nucleic Acids Res. 10: 2177–2187.

  3. 3

    Emtage, J.S., Angal, S., Doel, M.T., Harris, T.J.R., Jenkins, B., Lilley, G. and Lowe, P.A. 1983. Synthesis of calf prochymosin (prorennin) in Escherichia coli. Proc. Natl. Acad. Sci. USA. 80: 3671–3675.

  4. 4

    Harris, T.J.R. 1983. Expression of eukaryotic genes in E coli. p. 127–183. In: Genetic Engineering, vol. 4. Williamson, R., (ed.), Academic Press, London, U.K.

  5. 5

    Williams, D.C., Van Frank, R.M., Muth, W.L. and Burnett, J.P. 1982. Cytoplasmic inclusion bodies in E coli producing biosynthetic human insulin proteins. Science 215: 687–689.

  6. 6

    Cheng, Y.-S.E. (1983). Increased cell buoyant densities of protein overproducing E. coli cells. Biochem. Biophys. Res. Comm. 111: 104–111.

  7. 7

    Schoemaker, J.M., Brasnett, A.H. and Marston, F.A.O. 1984. Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies. Submitted.

  8. 8

    Roscnbusch, J.P. 1974. Characterization of the major envelope protein from Escherichia coli Regular arrangement on the peptidoglycan and unusual dodecyl sulphate binding. J. Biol. Chem. 249: 8019–8029.

  9. 9

    Schnaitman, C.A. 1971. Effect of elhylenediaminetetraaceticacid, Triton X-100 and Lysozyme on the morphology and chemical composition of isolated cell walls of Escherichia coli. J. Bacteriol. 108: 553–563.

  10. 10

    Goeddel, D.V., Heynecker, H.L., Hozumi, T., Arentzen, R., Itakura, K., Yansura, D.G., Ross, M.J., Miozzari, G., Crea, R. and Seeburg, P.H. 1979. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 281: 544–548.

  11. 11

    De Maeyer, E., Skup, D., Prasad, K.S.N., De Maeyer-Guignard, J., Williams, B., Sharpe, G., Pioli, D., Hennam, J., Schuch, W. and Atherton, K.T. 1982. Expression of a chemically synthesized a interferon gene. Proc. Natl. Acad. Sci. U.S.A. 79: 4256–4259.

  12. 12

    Foltmann, B., Pedersen, V.B., Jacobson, H., Kauffman, D. and Wybrandt, D. 1977. The complete amino acid sequence of prochymosin. Proc. Natl. Acad. Sci. U.S.A., 74: 2321–2324.

  13. 13

    Freedman, R.B. and Hillson, D.A. 1980. Formation of Disulphide Bonds, p. 157–212 In: The Enzymology of Post-translational Modification of Proteins, vol. I. Freedman, R. B. and Hawkins, H. G. (eds.), Academic Press, N.Y.

  14. 14

    Torchinski, Y.M. 1974. Sulphydryl and Disulphide Groups of Proteins, Plenum Press, New York.

  15. 15

    In: Data for Biochemical Research (2nd edition), 1969. p. 20–21. Dawson, R.M. G., Elliot, D.G., Elliot, W.H., and Jones, K.M. (eds.), Clarendon Press, Oxford.

  16. 16

    Boyer, H.W. and Roulland-Dussoix, D. 1969. Complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41: 459–472.

  17. 17

    Foltmann, B. 1970. Prochymosin and Chymosin (Prorennin and Rennin). Methods Enzymol. 19: 421–436.

  18. 18

    Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963). The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89: 114–123.

  19. 19

    Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analyl. Biochem. 72: 248–254.

  20. 20

    Read, S.M. and Northcote, D.H. 1981. Minimization of variation in the response to different proteins of the coomassie blue G dye-binding assay for protein. Analyt. Biochem. 116: 53–64.

  21. 21

    In: Handbook of Biochemistry and Molecular Biology (3rd edition), vol. III, 1976, p. 20. Fasman, G. D., (ed.), CRC Press, Inc.

  22. 22

    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of baeleriophagc T4. Nature (London) 227: 680–685.

  23. 23

    Towbin, H., Staehlin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350–4354.

  24. 24

    Burnette, W.N. 1981. ‘Western blotting’: electrophorelic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radio iodinated protein A. Analyt. Biochem. 112: 195–203.

Download references

Author information

Author notes

    • Joyce M. Schoemaker

    Present address: Department of Microbiology, The University of Chicago, Cummings Life Science Center, 920 East 58th Street, Chicago, Illinois, 60637, U.S.A.


  1. Department of Fermentation Development and Downstream Processing, Celltech Ltd., 250 Bath Road, Slough, SL1 4DY, Berkshire, U.K.

    • Fiona A. O. Marston
    • , Peter A. Lowe
    • , Susan White
    •  & Sarojani Angal
  2. Department of Molecular Biology, Celltech Ltd., 250 Bath Road, Slough, SL1 4DY, Berkshire, U.K.

    • Michael T. Doel


  1. Search for Fiona A. O. Marston in:

  2. Search for Peter A. Lowe in:

  3. Search for Michael T. Doel in:

  4. Search for Joyce M. Schoemaker in:

  5. Search for Susan White in:

  6. Search for Sarojani Angal in:

About this article

Publication history



Issue Date


Further reading