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Effect of Escherichia coli chaperonin GroELS on heterologously expressed human immunodeficiency virus type 1 reverse transcriptase in vivo and in vitro

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Abstract

The two subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (HIV-1 RT), p66 and p51, were coexpressed in Escherichia coli along with the E. coli chaperonin system GroEL/GroES. Coexpression increases the yield of heterodimeric HIV-1 RT by a factor of 4 to 5 and improves the nucleic acid binding affinity of HIV-1 RT by a factor of 1.6. We have analyzed the reasons for the improvements. The total increase in yield of HIV-1 RT can be attributed to an accumulation of RT subunits in the cells (factor of about 2.8) and an increased growth of the E. coli cells (factor of about 1.4). One reason for the accumulation in the cells is an improved stability of HIV-1 RT subunits toward bacterial proteases. In vitro studies showed that the nucleic acid binding affinity of HIV-1 RT purified from cells that did not coexpress GroELS was stimulated by adding purified GroELS (approx 1.5-fold), whereas HIV-1 RT stemming from cells coexpressing GroELS was stimulated only marginally (approx 1.1-fold). The in vivo as well as the in vitro studies suggest that the chaperonin interacts with HIV-1 RT and therefore affects the folding process of HIV-1 RT.

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References

  1. Hartl, F. U. (1996), Nature 381, 571–580.

    Article  CAS  Google Scholar 

  2. Beissinger, M. and Buchner, J. (1998), Biol. Chem. 379, 245–259.

    CAS  Google Scholar 

  3. Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989), Nature 337, 44–47.

    Article  CAS  Google Scholar 

  4. Lee, S. C. and Olins, P. O. (1992), J. Biol. Chem. 267, 2849–2852.

    CAS  Google Scholar 

  5. Wynn, R. M., Davie, J. R., Cox, R. P., and Chuang, D. T. (1992), J. Biol. Chem. 267, 12,400–12,403.

    CAS  Google Scholar 

  6. Dale, G. E., Schönfeld, H.-J., Langen, H., and Stieger, M. (1994), Protein Eng. 7, 925–931.

    Article  CAS  Google Scholar 

  7. Amrein, K. E., Takacs, B., Stieger, M., Molnos, J., Flint, N. A., and Burn, P. (1995), Proc. Natl. Acad. Sci. USA 92, 1048–1052.

    Article  CAS  Google Scholar 

  8. Jeong, W., Shin, N.-K., and Shin, H.-C. (1997), Biotechnol. Lett. 19, 579–582.

    Article  CAS  Google Scholar 

  9. Machida, S., Yu, Y., Singh, S. P., Kim, J.-D., Hayashi, K., and Kawata, Y. (1998), FEMS Microbiol. Lett. 159, 41–46.

    CAS  Google Scholar 

  10. Skalka, A.-M. and Goff, S. P., eds. (1993), Reverse Transcriptase, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  11. Arts, E. J. and Le Grice, S. F. J. (1998), in Prog. Nucleic Acid Res., vol. 58, Moldave, K., ed., Academic Press, pp. 339–393.

  12. Lightfoote, M. M., Coligan, J. E., Folks, T. M., Fauci, A. S., Martin, M. A., and Venkatesan, S. (1986), J. Virol. 60, 771–775.

    CAS  Google Scholar 

  13. Stahlhut, M. W. and Olsen, D. B. (1996), Methods Enzymol. 275, 122–133.

    CAS  Google Scholar 

  14. Lowe, D. M., Bradley, C., Darby, G. K., Larder, B. A., Powell, K. L., Purifoy, D. J. M., Tisdale, M., and Stammers, D. K. (1988), Biochemistry 27, 8884–8889.

    Article  CAS  Google Scholar 

  15. Maier, G., Dietrich, U., Panhans, B., Schröder, B., Rübsamen-Waigmann, H., Cellai, L., Hermann, T., and Heumann, H. (1999), Eur. J. Biochem. 261, 10–18.

    Article  CAS  Google Scholar 

  16. Müller, B., Restle, T., Weiss, S., Gautel, M., Sczakiel, G., and Goody, R. S. (1989), J. Biol. Chem. 264, 13,975–13,978.

    Google Scholar 

  17. Jonckheere, H., De Vreese, K., Debeyser, Z., Vanderkerckhove, J., Balzarini, J., Desmyter, J., De Clercq, E., and Anné, J. (1996), J. Virol. Methods 61, 113–125.

    Article  CAS  Google Scholar 

  18. Schröder, B. (1993), PhD thesis, Heinrich-Heine-University, Düsseldorf, Germany.

    Google Scholar 

  19. Laemmli, U. K. (1970), Nature 227, 680–685.

    Article  CAS  Google Scholar 

  20. Chen, M. and Christen, P. (1997), Anal. Biochem. 246, 263, 264.

    Article  CAS  Google Scholar 

  21. Choi, J.-K., Yoon, S.-H., Hong, H.-Y., Choi, D.-K., and Yoo, G.-S. (1996), Anal. Biochem. 236, 82–84.

    Article  CAS  Google Scholar 

  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor University Press, Cold Spring Harbor, NY.

    Google Scholar 

  23. Gründemann, D. and Schömig, E. (1996), BioTechniques 21, 898–903.

    Google Scholar 

  24. Boyle, J. S. and Lew, A. M. (1995), Trends Genetics 11, 8.

    Article  CAS  Google Scholar 

  25. Fayet, O., Louarn, J.-M., and Georgopoulos, C. (1986), Mol. Gen. Genetics 202, 435–445.

    Article  CAS  Google Scholar 

  26. Stegmann, R., Manakova, E., Rößle, M., Heumann, H., Nieba-Axmann, S. E., Plückthun, A., Hermann, T., May, R. P., and Wiedemann, A. (1998), J. Struct. Biol. 121, 30–40.

    Article  CAS  Google Scholar 

  27. Koller, G., Graumann, W., Kramer, W., Sara, M., and Jungbauer, A. (1995), J. Chromatogr. B 664, 107–118.

    Article  CAS  Google Scholar 

  28. Wang, J. D., Michelitsch, M. D., and Weissman, J. S. (1998), Proc. Natl. Acad. Sci. USA 95, 12,163–12,168.

    CAS  Google Scholar 

  29. Yagi, T. (1990), Kansenshogaku Zasshi—J. Jpn. Assoc. Infect. Dis. 64, 847–853.

    CAS  Google Scholar 

  30. Le Grice, S. F. J. (1990), Methods Enzymol. 185, 201–214.

    Article  Google Scholar 

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Authors and Affiliations

  1. Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152, Martinsried, Germany

    Gottfried Maier, Elena Manakova & Hermann Heumann

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  1. Gottfried Maier
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  2. Elena Manakova
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  3. Hermann Heumann
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Correspondence to Hermann Heumann.

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Maier, G., Manakova, E. & Heumann, H. Effect of Escherichia coli chaperonin GroELS on heterologously expressed human immunodeficiency virus type 1 reverse transcriptase in vivo and in vitro. Appl Biochem Biotechnol 87, 103–115 (2000). https://doi.org/10.1385/ABAB:87:2:103

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  • DOI: https://doi.org/10.1385/ABAB:87:2:103

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