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Applied Microbiology and Biotechnology

, Volume 37, Issue 5, pp 609–614 | Cite as

Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli

  • Ursula Rinas
  • James E. Bailey
Applied Genetics and Regulation

Summary

Culture conditions favouring the simulataneous formation of soluble protein and inclusion bodies (IBs) were chosen for producing the cytoplasmic protein β-galactosidase or the periplasmic protein TEM-β-lactomase. Soluble and insoluble cell fractions of Escherichia coli producing either β-galactosidase or TEM-β-lactomase were analyzed by one- and two-dimensional gel electrophoresis and subsequent silver staining or immunodection of the recombinant protein. The results show that truncated fragments of the recombinant protein were not present in the soluble cell fraction but accumulate in the IB fraction. The presense of other cellular, non-plasmid-encoded proteins in IB preparations such as the outer membrane proteins OmpF, OmpC, and OmpA or the ribosomal subunit proteins L7/L12 was attributed to co-precipitation of cell-debris-associated components. Protein-folding enzymes were not detected in IB preprations. The specificity of in-vivo protein association in the formation of IBs and its implication on protein purification is discussed.

Keywords

Recombinant Protein Inclusion Body Cell Fraction Compositional Analysis Outer Membrane Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Bowden GA, Georgiou G (1990) Folding and aggregation of β-lactamase in the periplasmic space of Escherichia coli. J Biol Chem 265:16760–16766Google Scholar
  2. Bowden GA, Paredes AM, Georgiou G (1991) Structure and morphology of protein inclusion bodies in Escherichia coli. Bio/ Technology 9:725–730Google Scholar
  3. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207Google Scholar
  4. Chalmers JJ, Kim E, Telford JN, Wong EY, Tacon WC, Shuler ML, Wilson DB (1990) Effects of temperature on Escherichia coli overproducing β-lactamase or human epidermal growth factor. Appl Environ Microbiol 56:104–111Google Scholar
  5. Cheng Y-SE (1983) Increased cell buoyant densities of protein overproducing Escherichia coli cells. Biochem Biophys Res Commun 111:104–111Google Scholar
  6. Garg VK, Costello MAC, Czuba BA (1991) Purification and production of therapeutic grade proteins. In: Seetharam R, Sharma SK (eds) Purification and analysis of recombinant proteins. Dekker, New York, pp 29–54Google Scholar
  7. Georgiou G, Baneyx F (1990) Expression, purification and immobilisation of a protein-A-β-lactamase hybrid protein. Ann N Y Acad Sci 589:139–147Google Scholar
  8. Georgiou G, Telford JN, Shuler ML, Wilson DB (1986) Localization of inclusion bodies in Escherichia coli overproducing β-lactamase or alkaline phosphatase. Appl Environ Microbiol 52:1157–1161Google Scholar
  9. Haase-Pettingell CA, King J (1988) Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation. A model for inclusion body formation. J Biol Chem 263:4977–4983Google Scholar
  10. Hart RA, Rinas U, Bailey JE (1990) Protein composition of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli. J Biol Chem 265:12728–12733Google Scholar
  11. Hartley DL, Kane JF (1988) Properties of inclusion bodies from recombinant Escherichia coli. Biochem Soc Trans 16:101–102Google Scholar
  12. Hochstrasser DF, Harrington MG, Hochstrasser A-C, Miller MJ, Merril CR (1988) Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal Biochem 173:424–435Google Scholar
  13. Kane JF, Hartley DL (1988) Formation of recombinant protein inclusion bodies in Escherichia coli. Trends Biotechnol 6:95–101Google Scholar
  14. Kane JF, Hartley DL (1991) Properties of recombinant protein-containing inclusion bodies in Escherichia coli. In: Seetharam R, Sharma SK (eds) Purification and analysis of recombinant proteins. Dekker, New York, pp 121–145Google Scholar
  15. Khosla C, Bailey JE (1989) Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli. J Bacteriol 171:5995–6004Google Scholar
  16. Kiefhaber T, Rudolph R, Kohler H-H, Buchner J (1991) Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding and aggregation. Bio/ Technology 9:825–829Google Scholar
  17. Kopetzki E, Schumacher G, Buckel P (1989) Control of formation of active soluble or inactive insoluble baker's yeast α-glucosidase PI in Escherichia coli by induction and growth conditions. Mol Gen Genet 216:149–155Google Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  19. Lin S, Zabin I (1972) β-Galactosidase: rates of synthesis and degradation of incomplete chains. J Biol Chem 147:2205–2211Google Scholar
  20. Marston FAO (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J 240:1–12Google Scholar
  21. Minsky A, Summers RG, Knowles JR (1986) Secretion of β-lactamase into the periplasm of Escherichia coli: evidence for a distinct release step associated with a conformational change. Proc Natl Acad Sci USA 83:4180–4184Google Scholar
  22. Mitraki A, King J (1989) Protein folding intermediates and inclusion body formation. Bio/Technology 7:690–697Google Scholar
  23. Mitzukami T, Komatsu Y, Hosoi N, Itoh S, Oka T (1986) Production of active human interferon-β in E. coli. I. Preferential production by lower culture temperature. Biotechnol Lett 8:605–610Google Scholar
  24. O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021Google Scholar
  25. Phillips TA, Vaughn V, Bloch PL, Neidhardt FC (1987) Gene-protein index of Escherichia coli K-12, Edition 2. In: Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium. ASM, Washington, D. C., pp 919–966Google Scholar
  26. Piatak M, Lane JA, Laird W, Bjorn MJ, Wang A, Williams M (1988) Expression of soluble and fully functional ricin A chain in Escherichia coli is temperature-sensitive. J Biol Chem 263:4837–4843Google Scholar
  27. Prouty WF, Karnovsky MJ, Goldberg AL (1975) Degradation of abnormal proteins in Escherichia coli. Formation of protein inclusions in cells exposed to amino acid analogs. J Biol Chem 250:1112–1122Google Scholar
  28. Schein CH (1989) Production of soluble recombinant proteins in bacteria. Bio/Technology 7:1141–1149Google Scholar
  29. Schein CH, Noteborn MHM (1988) Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/Technology 6:291–294Google Scholar
  30. Schoner RG, Ellis LF, Schoner BE (1985) Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/Technology 3:151–154Google Scholar
  31. Strandberg L, Enfors S-O (1991) Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli. Appl Environ Microbiol 57:1669–1674Google Scholar
  32. Sugimoto S, Yokoo Y, Hatakeyama N, Yotsuji A, Teshiba S, Hagino H (1991) Higher culture pH is preferable for inclusion body formation of recombinant salmon growth hormone in Escherichia coli. Biotechnol Lett 13:385–388Google Scholar
  33. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354PubMedGoogle Scholar
  34. VanBogelen RA, Hutton ME, Neidhardt FC (1990) Gene-protein database of Escherichia coli K-12: Edition 3. Electrophoresis 11:1131–1166Google Scholar
  35. Williams DC, Frank RM van, Muth WL, Burnett JP (1982) Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science 215:687–689Google Scholar
  36. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Ursula Rinas
    • 1
  • James E. Bailey
    • 1
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadenaUSA

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