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High-level expression of murine terminal deoxynucleotidyl transferase inEscherichia coli grown at low temperature and overexpressingargU tRNA

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Abstract

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5′-triphosphates onto the 3′-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production inEscherichia coli of murine TdT with minimal proteolysis and high specific activity.

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References

  1. Bollum, F. J. (1960) Calf thymus polymerase.J. Biol. Chem. 235, 2399–2403.

    PubMed  CAS  Google Scholar 

  2. Chang, L. M. S. and Bollum, F. J. (1971) Deoxynucleotide-polymerizing enzymes of calf thymus gland. IV. Homogeneous terminal deoxynucleotidyl transferase.J. Biol. Chem. 246, 909–916.

    PubMed  CAS  Google Scholar 

  3. Bollum, F. J. (1974) Terminal deoxynucleotidyl transferase, inThe Enzymes (Boyer, P. D., ed.), Academic Press, New York, pp. 145–171.

    Google Scholar 

  4. Coleman, M. S. (1977) Terminal deoxynucleotidyl transferase: characterization of extraction and assay conditions from human and calf tissue.Arch. Biochem. Biophys. 182, 525–532.

    Article  PubMed  CAS  Google Scholar 

  5. Chang, L. M. S. and Bollum, F. J. (1971) Enzymatic synthesis of oligodeoxynucleotides.Biochemistry 10, 536–542.

    Article  PubMed  CAS  Google Scholar 

  6. Goldschneider, I., Gregoire, K. E., Barton, R. A., and Bollum, F. J. (1977) Demonstration of terminal deoxynucleotidyl transferase in thymocytes by immunofluorescence.Proc. Natl. Acad. Sci. USA 74, 734–738.

    Article  PubMed  CAS  Google Scholar 

  7. Grégoire, K. E., Goldschneider, I., and Barton, R. W. (1977) Intracellular distribution of terminal deoxynucleotidyl transferase in rat bone marrow and thymus.Proc. Natl. Acad. Sci. USA 74, 3993–3996.

    Article  PubMed  Google Scholar 

  8. Bollum, F. J. (1979) Terminal deoxynucleotidyl transferase as a hemopoietic cell marker.Blood 54, 1203–1215.

    PubMed  CAS  Google Scholar 

  9. Grégoire, K. E., Goldschneider, I., Barton, R. W., and Bollum, F. J. (1979) Ontogeny of terminal deoxynucleotidyl transferase-positive cells in lymphohemopoietic tissues of rat and mouse.J. Immunol. 123, 1347–1352.

    PubMed  Google Scholar 

  10. Alt, F. W. and Baltimore, D. (1982) Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions.Proc. Natl. Acad. Sci. USA 79, 4118–4122.

    Article  PubMed  CAS  Google Scholar 

  11. Kallenbach, S., Goodhardt, M., and Rougeon, F. (1990) A rapid test for V(D)J recombinase activity.Nucleic Acids Res. 18, 6730.

    Article  PubMed  CAS  Google Scholar 

  12. Komori, T., Okada, A., Stewart, V., and Alt, F. W. (1993) Lack of N regions in antigen receptor variable region genes of TdT-deficient mice.Science 261, 1171–1175.

    Article  PubMed  CAS  Google Scholar 

  13. Gilfillan, S., Dierich, A., Lemeur, M., Benoist, C., and Mathis, D. (1993) Mice lacking TdT: mature animals with an immature lymphocyte repertoire.Science 261, 1175–1178.

    Article  PubMed  CAS  Google Scholar 

  14. Lewis, S. M. (1994) The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses.Adv. Immunol. 56, 27–150.

    PubMed  CAS  Google Scholar 

  15. Yang, B., Gathy, K. N., and Coleman, M. S. (1994) Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase.J. Biol. Chem. 269, 11,859–11,868.

    CAS  Google Scholar 

  16. Gilfillan, S., Bachmann, M., Trembleau, S., Adorini, L., Kalinke, U., Zinkernagel, R., et al. (1995) Efficient immune response in mice lacking N-region diversity.Eur. J. Immunol. 25, 3115–3122.

    Article  PubMed  CAS  Google Scholar 

  17. Bentolila, L. A., Wu, G. E., Nourrit, F., Fanton d’Andon, M., Rougeon, F., and Doyen, N. (1997) Constitutive expression of terminal deoxynucleotidyl transferase in transgenic mice is sufficient for N region diversity to occur at any Ig locus throughout B cell differentiation.J. Immunol. 158, 715–723.

    PubMed  CAS  Google Scholar 

  18. Peterson, R. C., Cheung, L. C., Mattaliano, R. J., Chang, L. M. S., and Bollum, F. J. (1984) Molecular cloning of human terminal deoxynucleotidyltransferase.Proc. Natl. Acad. Sci. USA 81, 4363–4367.

    Article  PubMed  CAS  Google Scholar 

  19. Koiwai, O., Yokota, T., Kageyama, T., Hirose, T., Yoshida, S., and Arai, K.-I. (1986) Isolation and characterization of bovine and mouse terminal deoxynucleotidyl transferase cDNAs expressible in mammalian cells.Nucleic Acids Res. 14, 5777–5792.

    Article  PubMed  CAS  Google Scholar 

  20. Lee, A. and Sue, E. (1994) Isolation and characterisation of the Xenopus terminal transferase.J. Immunol. 152, 4500–4507.

    PubMed  CAS  Google Scholar 

  21. Yang, B., Gathy, K. N., and Coleman, M. S. (1995) T-cell specific avian TdT: characterization of the cDNA and recombinant enzyme.Nucleic Acids Res. 23, 2041–2048.

    Article  PubMed  CAS  Google Scholar 

  22. Hansen, J. D. (1997) Characterization of rainbow trout terminal deoxynucleotidyl transferase structure and expression. TdT and Rag1 co-expression define the trout primary lymphoid tissues.Immunogenetics 46, 367–375.

    Article  PubMed  CAS  Google Scholar 

  23. Ito, J. and Braithwaite, D. K. (1991) Compilation and alignment of DNA polymerase sequences.Nucleic Acids Res. 19, 4045–4057.

    Article  PubMed  CAS  Google Scholar 

  24. Delarue, M., Poch, O., Tordo, N., Moras, D., and Argos, P. (1990) An attempt to unify the structure of polymerases.Protein Eng. 3, 461–467.

    Article  PubMed  CAS  Google Scholar 

  25. Holm, L. and Sander, C. (1995) DNA polymerase β belongs to an ancient nucleotidyltransferase superfamily.Trends Biochem. Sci. 20, 345–347.

    Article  PubMed  CAS  Google Scholar 

  26. Martin, G. and Keller, W. (1996) Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and a catalytic domain, homologous to the family X polymerases, and to other nucleotidyltransferases.EMBO J. 15, 2593–2603.

    PubMed  CAS  Google Scholar 

  27. Deibel, M. R. and Coleman, M. S. (1980) Limited proteolysis of calf thymus terminal deoxynucleotidyl transferase.Arch. Biochem. Biophys. 202, 414–419.

    Article  PubMed  CAS  Google Scholar 

  28. Chang, L. M. S., Plevani, P., and Bollum, F. J. (1982) Proteolytic degradation of calf thymus terminal deoxynucleotidyl transferase.J. Biol. Chem. 257, 5700–5706.

    PubMed  CAS  Google Scholar 

  29. Deibel, M. R. and Coleman, M. S. (1980) Biochemical properties of purified human terminal deoxynucleotidyltransferase.J. Biol. Chem. 255, 4206–4212.

    PubMed  CAS  Google Scholar 

  30. Chang, L. M. S., Rafter, E., Rusquet-Valerius, R., Peterson, R. C., White, S. T., and Bollum, F. J. (1988) Expression and processing of recombinant human terminal transferase in the baculovirus system.J. Biol. Chem. 263, 12,509–12,513.

    CAS  Google Scholar 

  31. Peterson, R. C., Cheung, L. C., Mattaliano, R. J., White, S. T., Chang, L. M. S., and Bollum, F. J. (1985) Expression of human terminal deoxynucleotidyl transferase inEscherichia coli.J. Biol. Chem. 260, 10,495–10,502.

    CAS  Google Scholar 

  32. Doyen, N., Fanton d’Andon, M., Bentolila, L. A., NGuyen, Q. T., and Rougeon, F. (1993) Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase.Nucleic Acids Res. 21, 1187–1191.

    Article  PubMed  CAS  Google Scholar 

  33. Bentolila, L. A., Fanton d’Andon, M., Nguyen, Q. T., Martinez, O., Rougeon, F., and Doyen, N. (1995) The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization.EMBO J. 14, 4221–4229.

    PubMed  CAS  Google Scholar 

  34. Parsot, C. (1986) Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase andd-serine dehydratase.EMBO J. 5, 3013–3019.

    PubMed  CAS  Google Scholar 

  35. Gill, A. B. C. and vonHippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data.Anal. Biochem. 182, 319–326.

    Article  PubMed  CAS  Google Scholar 

  36. Deibel, M. R. and Coleman, M. S. (1979) Purification of a high molecular weight human terminal deoxynucleotidyl transferase.J. Biol. Chem. 254, 8634–8640.

    PubMed  CAS  Google Scholar 

  37. Coleman, M. S. and Deibel, M. R. (1983) Terminal deoxynucleotidyl transferase, inEnzymes of Nucleic Acid Synthesis and Modification: DNA Enzymes (Jacob, S. T., ed.), CRC, Boca Raton, FL, pp. 93–118.

    Google Scholar 

  38. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990)Methods Enzymol. 185, 60–89.

    Article  PubMed  CAS  Google Scholar 

  39. Hernan, R. A., Hui, H. L., Andracki, M. E., Noble, R. W., Sligar, S. G., Walder, J. A., et al. (1992) Human hemoglobin expression inEscherichia coli: importance of optimal codon usage.Biochemistry 31, 8619–8628.

    Article  PubMed  CAS  Google Scholar 

  40. Kane, J. F. (1995) Effects of rare codon cluster on high-level expression of heterologous proteins inEscherichia coli. Curr. Opinion Biotechnol. 494–500.

  41. Wada, K., Wada, Y., Ishibashi, F., and Ikemura, T. (1992) Codon usage tabulated from the genebank genetic sequence data.Nucleic Acids Res. 20, 2111–2118.

    PubMed  CAS  Google Scholar 

  42. Spanjaard, R. A., Chen, K., Walker, J. R., and Duin, J. v. (1990) Frameshift suppression at tandem AGA and AGG codons by cloned tRNA genes: assigning a codon to argU tRNA and T4 tRNAarg.Nucleic Acids Res. 18, 5031–5036.

    Article  PubMed  CAS  Google Scholar 

  43. Saxena, P. and Walker, J. R. (1992) Expression of argU, the Escherichia coli gene coding for a rare Arginine tRNA.J. Bacteriol. 174, 1956–1964.

    PubMed  CAS  Google Scholar 

  44. Kurland, C. and Gallant, J. (1996) Errors of heterologous protein expression.Curr. Opinion Biotechnol. 7, 489–493.

    Article  CAS  Google Scholar 

  45. Schein, C. H. (1989) Production of soluble recombinant proteins in bacteria.Bio/technology 7, 1141–1149.

    CAS  Google Scholar 

  46. Chang, L. M. S. and Bollum, F. J. (1990) Multiple roles of divalent cation in the terminal deoxynucleotidyltransferase reaction.J. Biol. Chem. 265, 17,436–17,440.

    CAS  Google Scholar 

  47. Wetzel, R. (1992) Principles of protein stability. Part 2-enhanced folding and stabilization of proteins by suppression of aggregation in vitro and in vivo, in Protein Engineering—A Practical Approach (Rees, A. R., Sternberg, M. J. E., and Wetzel, R., eds.) IRL Press at Oxford University Press, Oxford, UK, pp. 191–219.

    Google Scholar 

  48. LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant K. L., Schendel, P. F., and McCoy, J. M. (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in theE. coli cytoplasm.Bio/technology 11, 187–193.

    Article  PubMed  CAS  Google Scholar 

  49. Medin, J. A. and Coleman, M. S. (1992) Lack of significance of Cys227 and Cys234 in terminal deoxynucleotidyltransferase.J. Biol. Chem. 8, 5199–5201.

    Google Scholar 

  50. Difilippantonio, M. J., McMahan, C. J., Eastman, Q. M., Spanopoulou, E., and Schatz, D. G. (1996) Rag1 mediates signal sequence recognition and recruitment of Rag2 in V(D)J recombination.Cell 87, 253–262.

    Article  PubMed  CAS  Google Scholar 

  51. Hiom, K. and Gellert, M. (1997) A stable Rag1–Rag2-DNA complex that is active in V(D)J cleavage.Cell 88, 65–72.

    Article  PubMed  CAS  Google Scholar 

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  1. Catherine Papanicolaou

    Present address: Department of Microbiology and Immunology, Tulane Medical School, 14301 Tulane Avenue, 70112, New Orleans, LA

Authors and Affiliations

  1. Unité de Génétique et Biochimie du Développement, URA CNRS 1960, Départment d’Immunologie, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris, France

    Jean-Baptiste Boulé, Emmett Johnson, François Rougeon & Catherine Papanicolaou

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  1. Jean-Baptiste Boulé
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  2. Emmett Johnson
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Correspondence to Catherine Papanicolaou.

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Boulé, JB., Johnson, E., Rougeon, F. et al. High-level expression of murine terminal deoxynucleotidyl transferase inEscherichia coli grown at low temperature and overexpressingargU tRNA. Mol Biotechnol 10, 199–208 (1998). https://doi.org/10.1007/BF02740839

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