Journal of Microbiology and Biotechnology

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

Analysis of Factors Affecting the Periplasmic Production of Recombinant Proteins in Escherichia coli

  • Published : 2007.08.30
Download PDF
⟨ Previous Next ⟩

Abstract

Five fusion proteins between Z domains derived from Staphylococcal Protein A and Green Fluorescent Protein or Human Proinsulin were produced on the periplasm of Escherichia coli. The effects of the molecular weight and amino acid composition of the translocated peptide, culture medium composition, and growth phase of the bacterial culture were analyzed regarding the expression and periplasmic secretion of the recombinant proteins. It was found that secretion was not affected by the size of the translocated peptide (17-42 kDa) and that the highest periplasmic production values were obtained on the exponential phase of growth. Moreover, the highest periplasmic values were obtained in minimal medium, showing the relevance of the culture medium composition on secretion. In silico prediction analysis suggested that with respect to the five proteins used in this study, those that are prone to form ${\alpha}$-helix structures are more translocated to the periplasm.

Keywords

References

  1. Albano, R., L. Randers-Eichorn, Q. Chang, W. Bentley, and G. Rao. 1996. Quantitative measurement of green fluorescent protein expression. Biotechnol. Tech. 10: 953-958
  2. Andersen, D. C. and L. Krummen. 2002. Recombinant protein expression for therapeutic applications. Curr. Opin. Biotechnol. 13: 117-123 https://doi.org/10.1016/S0958-1669(02)00300-2
  3. Baneyx, F. 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10: 411-421 https://doi.org/10.1016/S0958-1669(99)00003-8
  4. Baneyx, F. and M. Mujacic. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22: 1399-1408 https://doi.org/10.1038/nbt1029
  5. Belin, D., L.-M. Guzman, S. Bost, M. Konakova, F. Silva, and J. Beckwith. 2004. Functional activity of eukaryotic signal sequences in Escherichia coli: The ovalbumin family of serine protease inhibitors. J. Mol. Biol. 335: 437-453 https://doi.org/10.1016/j.jmb.2003.10.076
  6. Chudakov, D. M., S. Lukyanov, and K. A. Lukyanov. 2005. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23: 605-613 https://doi.org/10.1016/j.tibtech.2005.10.005
  7. Combet, C., C. Blanchet, C. Geourjon, and G. Deleage. 2000. NPS@: Network protein sequence analysis. Trends Biochem. Sci. 25: 147-150 https://doi.org/10.1016/S0968-0004(99)01540-6
  8. Dedhia, N., R. Richins, A. Mesina, and W. Chen. 1997. Improvement in recombinant protein production in ppGppdeficient Escherichia coli. Biotechnol. Bioeng. 53: 379-386 https://doi.org/10.1002/(SICI)1097-0290(19970220)53:4<379::AID-BIT4>3.0.CO;2-K
  9. 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 https://doi.org/10.1128/jb.177.6.1497-1504.1995
  10. Fahnert, B., H. Lilie, and P. Neubauer. 2004. Inclusion bodies: Formation and utilisation. Adv. Biochem. Eng. Biotechnol. 89: 93-142
  11. 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 https://doi.org/10.1128/JB.182.14.4068-4076.2000
  12. Hoffmann, F. and U. Rinas. 2004. Stress induced by recombinant protein production in Escherichia coli. Adv. Biochem. Eng. Biotechnol. 89: 73-92
  13. Hoffmann, F., J. van den Heuvel, N. Zidek, and U. Rinas. 2004. Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb. Tech. 34: 235-241 https://doi.org/10.1016/j.enzmictec.2003.10.011
  14. 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 https://doi.org/10.1074/jbc.M002937200
  15. 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 https://doi.org/10.1128/JB.182.8.2163-2169.2000
  16. Khokhlova, O. V. and M. A. Nesmeyanova. 2004. Interdependent effects of the charge of the N-terminal region of the signal peptide, SecA, and SecB on secretion of alkaline phosphatase in Escherichia coli. Mol. Biol. (Mosk) 38: 239-246 https://doi.org/10.1023/B:MBIL.0000023740.66749.b0
  17. Koster, M., W. Bitter, and J. Tommassen. 2000. Protein secretion mechanisms in Gram-negative bacteria. Int. J. Med. Microbiol. 290: 325-331 https://doi.org/10.1016/S1438-4221(00)80033-8
  18. Mergulhão, F. and G. Monteiro. 2004. Secretion capacity limitations of the Sec pathway in Escherichia coli. J. Microbiol. Biotechnol. 14: 128-133
  19. 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 https://doi.org/10.1007/s00253-003-1232-8
  20. Mergulhao, 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 https://doi.org/10.1385/MB:19:3:239
  21. Mergulhão, F. J., M. A. Taipa, J. M. Cabral, and G. A. Monteiro. 2004. Evaluation of bottlenecks in proinsulin secretion by Escherichia coli. J. Biotechnol. 109: 31-43 https://doi.org/10.1016/j.jbiotec.2003.10.024
  22. Mergulhão, F. J. M. and G. A. Monteiro. 2005. Recombinant protein secretion in Escherichia coli. Biotechnol. Adv. 23: 177-202 https://doi.org/10.1016/j.biotechadv.2004.11.003
  23. Mergulhao, F. J. M., G. A. Monteiro, J. M. S. Cabral, and M. A. Taipa. 2004. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J. Microbiol. Biotechnol. 14: 1-14
  24. 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 https://doi.org/10.1042/BA20030043
  25. Moks, T., L. Abrahmsen, E. Holmgren, M. Bilich, A. Olsson, M. Uhlen, G. Pohl, C. Sterky, H. Hultberg, S. Josephson, et al. 1987. Expression of human insulin-like growth factor I in bacteria: Use of optimized gene fusion vectors to facilitate protein purification. Biochemistry 26: 5239-5244 https://doi.org/10.1021/bi00391a005
  26. Nakai, K. and M. Kanehisa. 1991. Expert system for predicting protein localization sites in Gram-negative bacteria. Proteins 11: 95-110 https://doi.org/10.1002/prot.340110203
  27. Neubauer, P., H. Y. Lin, and B. Mathiszik. 2003. Metabolic load of recombinant protein production: Inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli. Biotechnol. Bioeng. 83: 53-64 https://doi.org/10.1002/bit.10645
  28. 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 https://doi.org/10.1128/JB.183.4.1346-1358.2001
  29. Rosenberg, H. F. 1998. Isolation of recombinant secretory proteins by limited induction and quantitative harvest. Biotechniques 24: 188-191
  30. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd Ed. CSH Press, Cold Spring Harbour
  31. 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 https://doi.org/10.1002/bit.10457
  32. 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 https://doi.org/10.1128/jb.177.18.5238-5246.1995
  33. 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 https://doi.org/10.1038/nbt0596-629
  34. Stahl, S. and P. A. Nygren. 1997. The use of gene fusions to protein A and protein G in immunology and biotechnology. Pathol. Biol. (Paris) 45: 66-76
  35. 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 https://doi.org/10.1046/j.1365-2958.2002.02797.x
  36. Yokoyama, S. 2003. Protein expression systems for structural genomics and proteomics. Curr. Opin. Chem. Biol. 7: 39-43 https://doi.org/10.1016/S1367-5931(02)00019-4
  37. Zhang, J., R. E. Campbell, A. Y. Ting, and R. Y. Tsien. 2002. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3: 906-918 https://doi.org/10.1038/nrm976