Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Secretion Capacity Limitations of the Sec Pathway in Escherichia coli

  • Mergulhao, Filipe J.M. (Centro de Engenharia Biologica e Quimica, Institute Superior Tecnico) ;
  • Monteiro, Gabriel A. (Centro de Engenharia Biologica e Quimica, Institute Superior Tecnico)
  • Published : 2004.02.01
Download PDF
⟨ Previous Next ⟩

Abstract

The secretion capacity of two E. coli strains (JM109 and AF1000) was evaluated through the expression of two human proinsulin fusion proteins using the translocation signal sequence from Staphylococcal protein A (SpA). Although a 7 to 11-fold difference in the expression levels was attained by the use of different promoters (SpA and malK promoters) and copy-number vectors (700 and 50 copies per cell), the maximum translocation rates for all the systems were around 140,000 amino acids $cell^{-1} min^{-1}$. Moreover, the secretion capacity was found to be independent of the size of the exiting peptide and its translational rate.

Keywords

References

  1. Andersson, H. and G. von Heijne. 1991. A 30-residue-long “export initiation domain” adjacent to the signal sequence is critical for protein translocation across the inner membrane of Escherichia coli. Proc. Natl. Acad. Sci. USA 88: 9751- 9754
  2. Baneyx, F. 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10: 411-421
  3. Beck, K., L. F. Wu, J. Brunner, and M. Muller. 2000. Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor. EMBO J. 19: 134-143
  4. Behrmann, M., H. G. Koch, T. Hengelage, B. Wieseler, H. K. Hoffschulte, and M. Muller. 1998. Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli. J. Biol. Chem. 273: 13898-13904 https://doi.org/10.1074/jbc.273.22.13898
  5. Brendel, V., P. Bucher, I. R. Nourbakhsh, B. E. Blaisdell, and S. Karlin. 1992. Methods and algorithms for statistical analysis of protein sequences. Proc. Natl. Acad. Sci. USA 89: 2002-2006
  6. Breyton, C., W. Haase, T. A. Rapoport, W. Kuhlbrandt, and I. Collinson. 2002. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418: 662-665
  7. de Gier, J. W. and J. Luirink. 2001. Biogenesis of inner membrane proteins in Escherichia coli. Mol. Microbiol. 40: 314-322
  8. Dong, H., L. Nilsson, and C. G. Kurland. 1995. Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction. J. Bacteriol. 177: 1497-1504
  9. Economou, A. 1999. Following the leader: bacterial protein export through the Sec pathway. Trends Microbiol. 7: 315- 320
  10. Feilmeier, B. J., G. Iseminger, D. Schroeder, H. Webber, and G. J. Phillips. 2000. Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J. Bacteriol. 182: 4068-4076
  11. Fekkes, P. and A. J. Driessen. 1999. Protein targeting to the bacterial cytoplasmic membrane. Microbiol. Mol. Biol. Rev. 63: 161-173
  12. Guisez, Y., I. Fache, L. A. Campfield, F. J. Smith, A. Farid, G. Plaetinck, J. Van der Heyden, J. Tavernier, W. Fiers, P. Burn, and R. Devos. 1998. Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization. Protein. Expr. Purif. 12: 249-258
  13. Herskovits, A. A., E. S. Bochkareva, and E. Bibi. 2000. New prospects in studying the bacterial signal recognition particle pathway. Mol. Microbiol. 38: 927-939
  14. Hesterkamp, T., E. Deuerling, and B. Bukau. 1997. The amino-terminal 118 amino acids of Escherichia coli trigger factor constitute a domain that is necessary and sufficient for binding to ribosomes. J. Biol. Chem. 272: 21865-21871
  15. Hesterkamp, T., S. Hauser, H. Lutcke, and B. Bukau. 1996. Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains. Proc. Natl. Acad. Sci. USA 93: 4437-4441
  16. Hirst, T. R. and J. Holmgren. 1987. Conformation of protein secreted across bacterial outer membranes: A study of enterotoxin translocation from Vibrio cholerae. Proc. Natl. Acad. Sci. USA 84: 7418-7422
  17. Huang, H. C., M. Y. Sherman, O. Kandror, and A. L. Goldberg. 2001. The molecular chaperone DnaJ is required for the degradation of a soluble abnormal protein in Escherichia coli. J. Biol. Chem. 276: 3920-3928
  18. Joseleau-Petit, D., D. Vinella, and R. D'Ari. 1999. Metabolic alarms and cell division in Escherichia coli. J. Bacteriol. 181: 9-14
  19. Kajava, A. V., S. N. Zolov, A. E. Kalinin, and M. A. Nesmeyanova. 2000. The net charge of the first 18 residues of the mature sequence affects protein translocation across the cytoplasmic membrane of gram-negative bacteria. J. Bacteriol. 182: 2163-2169
  20. Kim, J., J. Luirink, and D. A. Kendall. 2000. SecB dependence of an exported protein is a continuum influenced by the characteristics of the signal peptide or early mature region. J. Bacteriol. 182: 4108-4112
  21. Koster, M., W. Bitter, and J. Tommassen. 2000. Protein secretion mechanisms in Gram-negative bacteria. Int. J. Med. Microbiol. 290: 325-331
  22. Lee, J., V. Saraswat, I. Koh, K. B. Song, Y. H. Park, and S. K. Rhee. 2001. Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli. FEMS Microbiol. Lett. 195: 127-132
  23. Makrides, S. C. 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60: 512-538
  24. Manting, E. H. and A. J. Driessen. 2000. Escherichia coli translocase: the unravelling of a molecular machine. Mol. Microbiol. 37: 226-238
  25. Mason, N., L. F. Ciufo, and J. D. Brown. 2000. Elongation arrest is a physiologically important function of signal recognition particle. EMBO J. 19: 4164-4174
  26. Mergulhão, F., G. Monteiro, A. Kelly, M. Taipa, and J. Cabral. 2000. Recombinant human proinsulin: A new approach in gene assembly and protein expression. J. Microbiol. Biotechnol. 10: 690-693
  27. Mergulhão, F., G. Monteiro, G. Larsson, A. Sandem, A. Farewell, T. Nystrom, J. Cabral, and M. Taipa. 2003. Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB. Appl. Microbiol. Biotechnol. 61: 495-501
  28. Mergulhão, F. J., A. G. Kelly, G. A. Monteiro, M. A. Taipa, and J. M. Cabral. 1999. Troubleshooting in gene splicing by overlap extension: A step-wise method. Mol. Biotechnol. 12: 285-287
  29. Mergulhão, F. J., G. A. Monteiro, J. M. Cabral, and M. A. Taipa. 2001. A quantitative ELISA for monitoring the secretion of ZZ-fusion proteins using SpA domain as immunodetection reporter system. Mol. Biotechnol. 19: 239-244
  30. Mergulhão, F. J. M., G. A. Monteiro, G. Larsson, M. Bostrom, A. Farewell, T. Nystrom, J. M. S. Cabral, and M. A. Taipa. 2003. Evaluation of inducible promoters on the secretion of a ZZ-Proinsulin fusion protein. Biotechnol. Appl. Biochem. 38: 87-93
  31. Miller, K. W., R. Schamber, Y. Chen, and B. Ray. 1998. Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon. Appl. Environ. Microbiol. 64: 14-20
  32. Missiakas, D. and S. Raina. 1997. Protein misfolding in the cell envelope of Escherichia coli: New signaling pathways. Trends Biochem. Sci. 22: 59-63
  33. Nakai, K. and M. Kanehisa. 1991. Expert system for predicting protein localization sites in gram-negative bacteria. Proteins 11: 95-110
  34. Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6
  35. Nishiyama, K., A. Fukuda, K. Morita, and H. Tokuda. 1999. Membrane deinsertion of SecA underlying proton motive force-dependent stimulation of protein translocation. EMBO J. 18: 1049-1058
  36. Nouwen, N. and A. J. Driessen. 2002. SecDFyajC forms a heterotetrameric complex with YidC. Mol. Microbiol. 44: 1397-1405
  37. Oliver, D., J. Norman, and S. Sarker. 1998. Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon. J. Bacteriol. 180: 5240-5242
  38. Page, N., D. Kluepfel, F. Shareck, and R. Morosoli. 1996. Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans. Appl. Environ. Microbiol. 62: 109-114
  39. Palacios, J. L., I. Zaror, P. Martinez, F. Uribe, P. Opazo, T. Socias, M. Gidekel, and A. Venegas. 2001. Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi. J. Bacteriol. 183: 1346- 1358
  40. Patzelt, H., S. Rudiger, D. Brehmer, G. Kramer, S. Vorderwulbecke, E. Schaffitzel, A. Waitz, T. Hesterkamp, L. Dong, J. Schneider-Mergener, B. Bukau, and E. Deuerling. 2001. Binding specificity of Escherichia coli trigger factor. Proc. Natl. Acad. Sci. USA 98: 14244-14249
  41. Prinz, W. A., F. Aslund, A. Holmgren, and J. Beckwith. 1997. The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J. Biol. Chem. 272: 15661-15667
  42. Pugsley, A. P. 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57: 50- 108
  43. Rosenberg, H. F. 1998. Isolation of recombinant secretory proteins by limited induction and quantitative harvest. Biotechniques 24: 188-191
  44. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. CSH Laboratory Press, Cold Spring Harbor, U.S.A
  45. Sandén, A. M., I. Prytz, I. Tubulekas, C. Forberg, H. Le, A. Hektor, P. Neubauer, Z. Pragai, C. Harwood, A. Picon, J. Teixeira de Mattos, P. Postma, A. Farewell, T. Nystrom, S. Reeh, S. Pedersen, and G. Larsson. 2003. Limiting factors in Escherichia coli fed-batch production of recombinant protein. Biotechnol. Bioeng. 81: 158-166
  46. Sarker, S. and D. Oliver. 2002. Critical regions of secM that control its translation and secretion and promote secretionspecific secA regulation. J. Bacteriol. 184: 2360-2369
  47. Sarker, S., K. E. Rudd, and D. Oliver. 2000. Revised translation start site for secM defines an atypical signal peptide that regulates Escherichia coli secA expression. J. Bacteriol. 182: 5592-5595
  48. Sauvonnet, N., I. Poquet, and A. P. Pugsley. 1995. Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end. J. Bacteriol. 177: 5238-5246
  49. Schmidt, M., E. Viaplana, F. Hoffmann, S. Marten, A. Villaverde, and U. Rinas. 1999. Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway. Biotechnol. Bioeng. 66: 61-67
  50. Simmons, L. C. and D. G. Yansura. 1996. Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli. Nat. Biotechnol. 14: 629-634
  51. Stanley, N. R., F. Sargent, G. Buchanan, J. Shi, V. Stewart, T. Palmer, and B. C. Berks. 2002. Behaviour of topological marker proteins targeted to the Tat protein transport pathway. Mol. Microbiol. 43: 1005-1021
  52. Summers, R. G., C. R. Harris, and J. R. Knowles. 1989. A conservative amino acid substitution, arginine for lysine, abolishes export of a hybrid protein in Escherichia coli. Implications for the mechanism of protein secretion. J. Biol. Chem. 264: 20082-20088
  53. Summers, R. G. and J. R. Knowles. 1989. Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein. J. Biol. Chem. 264: 20074-20081
  54. Swartz, J. R. 2001. Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 12: 195- 201 https://doi.org/10.1016/S0958-1669(00)00199-3
  55. Tan, N. S., B. Ho, and J. L. Ding. 2002. Engineering a novel secretion signal for cross-host recombinant protein expression. Protein Eng. 15: 337-345
  56. Tian, H. and J. Beckwith. 2002. Genetic screen yields mutations in genes encoding all known components of the Escherichia coli signal recognition particle pathway. J. Bacteriol. 184: 111-118
  57. Triplett, T. L., A. R. Sgrignoli, F. B. Gao, Y. B. Yang, P. C. Tai, and L. M. Gierasch. 2001. Functional signal peptides bind a soluble N-terminal fragment of SecA and inhibit its ATPase activity. J. Biol. Chem. 276: 19648-19655
  58. Valent, Q. A., P. A. Scotti, S. High, J. W. de Gier, G. von Heijne, G. Lentzen, W. Wintermeyer, B. Oudega, and J. Luirink. 1998. The Escherichia coli SRP and SecB targeting pathways converge at the translocon. EMBO J. 17: 2504- 2512
  59. van der Wolk, J. P., P. Fekkes, A. Boorsma, J. L. Huie, T. J. Silhavy, and A. J. Driessen. 1998. PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation. EMBO J. 17: 3631-3639
  60. Veenendaal, A. K., C. van der Does, and A. J. Driessen. 2001. Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis. J. Biol Chem. 276: 32559-32566
  61. Wang, L., A. Miller, and D. A. Kendall. 2000. Signal peptide determinants of SecA binding and stimulation of ATPase activity. J. Biol. Chem. 275: 10154-10159
  62. Yahr, T. L. and W. T. Wickner. 2000. Evaluating the oligomeric state of SecYEG in preprotein translocase. EMBO J. 19: 4393-4401