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Purification of Calf Prochymosin (Prorennin) Synthesized in Escherichia Coli

Abstract

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.

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References

  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.

    Chapter  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Google Scholar 

  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.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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. 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.

    Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Google Scholar 

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

  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.

    Google Scholar 

  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.

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

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

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

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

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Marston, F., Lowe, P., Doel, M. et al. Purification of Calf Prochymosin (Prorennin) Synthesized in Escherichia Coli. Nat Biotechnol 2, 800–804 (1984). https://doi.org/10.1038/nbt0984-800

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