Isolation and Purification of Protein Granules from Escherichia coli Cells Overproducing Bovine Growth Hormone

Abstract

High-level expression of bovine growth hormone in Escherichia coli results in the formation of distinct cytoplasmic granules that are visible with the phase-contrast microscope. These granules were examined by transmission and scanning electron microscopy. Intact granules were isolated from crude cell lysates by differential centrifugation and were further purified by a simple washing procedure that yields nearly homogeneous bovine growth hormone.

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References

  1. 1

    Gribskov, M. and Burgess, R.R. 1983. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene 26: 109–118.

  2. 2

    Masui, Y., Mizuno, T. and Inouye, M. 1984. Novel high-level expression cloning vehicles: 104-fold amplification of Escherichia coli minor protein. Bio/Technology 2 81–85.

  3. 3

    Ho, Y.-S., Lewis, M. and Rosenberg, M. 1982. Purification and properties of a transcriptional activator. J. Biol. Chem. 257: 9128–9134.

  4. 4

    Simons, G., Remaut, E., Allet, B., Devos, R. and Fiers, W. 1984. High-level expression of human interferon gamma in Escherichia coli under control of the PL promoter of bacteriophage lambda. Gene 28 55–64.

  5. 5

    Devos, R., Plaetinck, G., Cheroutre, H., Simons, G., Degrave, W., Tavernier, J., Remaut, E. and Fiers, W. 1983. Molecular cloning of human intereukin 2 cDNA and its expression in E. coli. Nucleic Acids Res. 11: 4307–4323.

  6. 6

    Cheng, Y.-S.E., Kwoh, D.Y., Kwoh, T.J., Soltvedt, B.C. and Zipser, D. 1981. Stabilization of a degradable protein by its Overexpression in Escherichia coli. Gene 14: 121–130.

  7. 7

    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.

  8. 8

    Kleid, D.G., Yansura, D., Small, B., Dowbenko, D., Moore, D.M., Grubman, M.J., McKercher, P.D., Morgan, D.O., Robertson, B.H. and Bachrach, H.L. 1981. Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science 214: 1125–1129.

  9. 9

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

  10. 10

    Paul, D.C., Van Frank, R.M., Muth, W.L., Ross, J.W. and Williams, D.C. 1983. Immunocytochemical demonstration of human proinsulin chimeric polypeptide within cytoplasmic inclusion bodies of Escherichia coli. European J. Cell Biol. 31: 171–174.

  11. 11

    Schoner, B.E., Hsiung, H.M., Belagaje, R.M., Mayne, N.G. and Schoner, R.G. 1984. Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli. Proc. Natl. Acad. Sci. (USA) 81: 5403–5407.

  12. 12

    Prouty, W.F. and Goldberg, A.L. 1972. Fate of abnormal proteins in E. coli accumulation in intracellular granules before catabolism. Nature New Biol. 240: 147–150.

  13. 13

    Prouty, W.F., Karnovsky, M.J. and Goldberg, A.L. 1975. Degradation of abnormal proteins in Escherichia coli. J. Biol. Chem. 250: 1112–1123.

  14. 14

    Millonig, G. 1961. Advantages of a phosphate buffer for osmium tetraoxide solutions in fixation. J. Appl. Phys. 32: 1637–1639.

  15. 15

    Spurr, A.R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastructure Research 26: 31–36.

  16. 16

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

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Schoner, R., Ellis, L. & Schoner, B. Isolation and Purification of Protein Granules from Escherichia coli Cells Overproducing Bovine Growth Hormone. Nat Biotechnol 3, 151–154 (1985). https://doi.org/10.1038/nbt0285-151

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