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. 1990 Jul;87(14):5573–5577. doi: 10.1073/pnas.87.14.5573

Isolation of mutants of human immunodeficiency virus protease based on the toxicity of the enzyme in Escherichia coli.

E Z Baum 1, G A Bebernitz 1, Y Gluzman 1
PMCID: PMC54367  PMID: 2196572

Abstract

The protease encoded by the pol gene of human immunodeficiency virus was expressed in Escherichia coli and found to be toxic to strain BL21(DE3). This toxicity provided a convenient selection for isolating mutants of the protease that are nontoxic and enzymatically inactive. This strong correlation between functional protease and toxicity resulted in rapid identification of several protease mutations, including mutations that exhibit temperature sensitivity. A total of 24 missense mutations and 7 nonsense mutations were identified. The described selection procedure may have wider applications for isolating mutants of other eukaryotic proteins that exhibit a toxic phenotype in E. coli.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Billich S., Knoop M. T., Hansen J., Strop P., Sedlacek J., Mertz R., Moelling K. Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease. J Biol Chem. 1988 Dec 5;263(34):17905–17908. [PubMed] [Google Scholar]
  2. Darke P. L., Leu C. T., Davis L. J., Heimbach J. C., Diehl R. E., Hill W. S., Dixon R. A., Sigal I. S. Human immunodeficiency virus protease. Bacterial expression and characterization of the purified aspartic protease. J Biol Chem. 1989 Feb 5;264(4):2307–2312. [PubMed] [Google Scholar]
  3. Darke P. L., Nutt R. F., Brady S. F., Garsky V. M., Ciccarone T. M., Leu C. T., Lumma P. K., Freidinger R. M., Veber D. F., Sigal I. S. HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins. Biochem Biophys Res Commun. 1988 Oct 14;156(1):297–303. doi: 10.1016/s0006-291x(88)80839-8. [DOI] [PubMed] [Google Scholar]
  4. Debouck C., Gorniak J. G., Strickler J. E., Meek T. D., Metcalf B. W., Rosenberg M. Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8903–8906. doi: 10.1073/pnas.84.24.8903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Farmerie W. G., Loeb D. D., Casavant N. C., Hutchison C. A., 3rd, Edgell M. H., Swanstrom R. Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. Science. 1987 Apr 17;236(4799):305–308. doi: 10.1126/science.2436298. [DOI] [PubMed] [Google Scholar]
  6. Fowler R. G., Degnen G. E., Cox E. C. Mutational specificity of a conditional Escherichia coli mutator, mutD5. Mol Gen Genet. 1974;133(3):179–191. doi: 10.1007/BF00267667. [DOI] [PubMed] [Google Scholar]
  7. Graves M. C., Lim J. J., Heimer E. P., Kramer R. A. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2449–2453. doi: 10.1073/pnas.85.8.2449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hansen J., Billich S., Schulze T., Sukrow S., Moelling K. Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody. EMBO J. 1988 Jun;7(6):1785–1791. doi: 10.1002/j.1460-2075.1988.tb03009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kohl N. E., Emini E. A., Schleif W. A., Davis L. J., Heimbach J. C., Dixon R. A., Scolnick E. M., Sigal I. S. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686–4690. doi: 10.1073/pnas.85.13.4686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kräusslich H. G., Ingraham R. H., Skoog M. T., Wimmer E., Pallai P. V., Carter C. A. Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides. Proc Natl Acad Sci U S A. 1989 Feb;86(3):807–811. doi: 10.1073/pnas.86.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kräusslich H. G., Schneider H., Zybarth G., Carter C. A., Wimmer E. Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli. J Virol. 1988 Nov;62(11):4393–4397. doi: 10.1128/jvi.62.11.4393-4397.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lapatto R., Blundell T., Hemmings A., Overington J., Wilderspin A., Wood S., Merson J. R., Whittle P. J., Danley D. E., Geoghegan K. F. X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature. 1989 Nov 16;342(6247):299–302. doi: 10.1038/342299a0. [DOI] [PubMed] [Google Scholar]
  13. Le Grice S. F., Mills J., Mous J. Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation. EMBO J. 1988 Aug;7(8):2547–2553. doi: 10.1002/j.1460-2075.1988.tb03103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Loeb D. D., Hutchison C. A., 3rd, Edgell M. H., Farmerie W. G., Swanstrom R. Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases. J Virol. 1989 Jan;63(1):111–121. doi: 10.1128/jvi.63.1.111-121.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Loeb D. D., Swanstrom R., Everitt L., Manchester M., Stamper S. E., Hutchison C. A., 3rd Complete mutagenesis of the HIV-1 protease. Nature. 1989 Aug 3;340(6232):397–400. doi: 10.1038/340397a0. [DOI] [PubMed] [Google Scholar]
  16. Meek T. D., Dayton B. D., Metcalf B. W., Dreyer G. B., Strickler J. E., Gorniak J. G., Rosenberg M., Moore M. L., Magaard V. W., Debouck C. Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1841–1845. doi: 10.1073/pnas.86.6.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Moffatt B. A., Studier F. W. T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell. 1987 Apr 24;49(2):221–227. doi: 10.1016/0092-8674(87)90563-0. [DOI] [PubMed] [Google Scholar]
  18. Moore M. L., Bryan W. M., Fakhoury S. A., Magaard V. W., Huffman W. F., Dayton B. D., Meek T. D., Hyland L., Dreyer G. B., Metcalf B. W. Peptide substrates and inhibitors of the HIV-1 protease. Biochem Biophys Res Commun. 1989 Mar 15;159(2):420–425. doi: 10.1016/0006-291x(89)90008-9. [DOI] [PubMed] [Google Scholar]
  19. Navia M. A., Fitzgerald P. M., McKeever B. M., Leu C. T., Heimbach J. C., Herber W. K., Sigal I. S., Darke P. L., Springer J. P. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature. 1989 Feb 16;337(6208):615–620. doi: 10.1038/337615a0. [DOI] [PubMed] [Google Scholar]
  20. Pearl L. H., Taylor W. R. A structural model for the retroviral proteases. Nature. 1987 Sep 24;329(6137):351–354. doi: 10.1038/329351a0. [DOI] [PubMed] [Google Scholar]
  21. Plotch S. J., Palant O., Gluzman Y. Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli. J Virol. 1989 Jan;63(1):216–225. doi: 10.1128/jvi.63.1.216-225.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Seelmeier S., Schmidt H., Turk V., von der Helm K. Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6612–6616. doi: 10.1073/pnas.85.18.6612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  24. Wlodawer A., Miller M., Jaskólski M., Sathyanarayana B. K., Baldwin E., Weber I. T., Selk L. M., Clawson L., Schneider J., Kent S. B. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science. 1989 Aug 11;245(4918):616–621. doi: 10.1126/science.2548279. [DOI] [PubMed] [Google Scholar]

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