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Functional expression of trypsin from Streptomyces griseus by Pichia pastoris

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6 U ml−1 (batch cultivation with flask cultivation) or 14.4 U ml−1 (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.

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References

  1. Bergmeyer HU, Gawehn K (1974) In: Bergmeyer HU (ed) Methods of enzymatic analysis, 2nd edn, vol 1. Academic, New York, pp 1013–1018

  2. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  PubMed  CAS  Google Scholar 

  3. Chi WJ, Song JH, Oh EA, Park SW, Chang YK, Kim ES, Hong SK (2009) Medium optimization and application of an affinity column chromatography for Streptomyces griseus trypsin production from the recombinant Streptomyces griseus. J Microbiol Biotechnol 19:1191–1196

    Article  PubMed  CAS  Google Scholar 

  4. Choi SS, Chi WJ, Lee JH, Kang SS, Jeong BC, Hong SK (2001) Overexpression of the sprD gene encoding Streptomyces griseus protease D stimulates actinorhodin production in Streptomyces lividans. J Microbiol 39:305–313

    CAS  Google Scholar 

  5. Corey DR, Shiau AK, Yang Q, Janowski BA, Craik CS (1993) Trypsin display on the surface of bacteriophage. Gene 128:129–134

    Article  PubMed  CAS  Google Scholar 

  6. Craik CS, Largman C, Fletcher T, Roczniak S, Barr PJ, Fletterick R, Rutter WJ (1985) Redesigning trypsin: alteration of substrate specificity. Science 228:291–297

    Article  PubMed  CAS  Google Scholar 

  7. Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52

    Article  PubMed  CAS  Google Scholar 

  8. Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138

    Article  PubMed  CAS  Google Scholar 

  9. Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278

    Article  PubMed  CAS  Google Scholar 

  10. Ghosalkar A, Sahai V, Srivastava A (2008) Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production. Bioresour Technol 99:7906–7910

    Article  PubMed  CAS  Google Scholar 

  11. Hanquier J, Sorlet Y, Desplancq D, Baroche L, Ebtinger M, Lefevre JF, Pattus F, Hershberger CL, Vertes AA (2003) A single mutation in the activation site of bovine trypsinogen enhances its accumulation in the fermentation broth of the yeast Pichia pastoris. Appl Environ Microbiol 69:1108–1113

    Article  PubMed  CAS  Google Scholar 

  12. Hartner FS, Glieder A (2006) Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Fact 5:39

    Article  PubMed  Google Scholar 

  13. Hohenblum H, Vorauer-Uhl K, Katinger H, Mattanovich D (2004) Bacterial expression and refolding of human trypsinogen. J Biotechnol 109:3–11

    Article  PubMed  CAS  Google Scholar 

  14. Horinouchi S (2002) A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus. Front Biosci 7:2045–2057

    Article  Google Scholar 

  15. Jungo C, Rerat C, Marison IW, von Stockar U (2006) Quantitative characterization of the regulation of the synthesis of alcohol oxidase and of the expression of recombinant avidin in a Pichia pastoris Mut(+) strain. Enzyme Microb Tech 39:936–944

    Article  CAS  Google Scholar 

  16. Kato JY, Chi WJ, Ohnishi Y, Hong SK, Horinouchi S (2005) Transcriptional control by A-factor of two trypsin genes in Streptomyces griseus. J Bacteriol 187:286–295

    Article  PubMed  CAS  Google Scholar 

  17. Kim JC, Cha SH, Jeong ST, Oh SK, Byun SM (1991) Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene. Biochem Biophys Res Commun 181:707–713

    Article  PubMed  CAS  Google Scholar 

  18. Kim NS, Yu HY, Chung ND, Shin YJ, Kwon TH, Yang MS (2011) Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures. Protein Expr Purif 76:121–126

    Article  PubMed  CAS  Google Scholar 

  19. Kossiakoff AA, Chambers JL, Kay LM, Stroud RM (1977) Structure of bovine trypsinogen at 1.9 Å resolution. Biochemistry 16:654–664

    Article  PubMed  CAS  Google Scholar 

  20. Macouzet M, Simpson BK, Lee BH (2005) Expression of a cold-adapted fish trypsin in Pichia pastoris. FEMS Yeast Res 5:851–857

    Article  PubMed  CAS  Google Scholar 

  21. Niles AL, Maffitt M, Haak-Frendscho M, Wheeless CJ, Johnson DA (1998) Recombinant human mast cell tryptase beta: stable expression in Pichia pastoris and purification of fully active enzyme. Biotechnol Appl Biochem 28:125–131

    PubMed  CAS  Google Scholar 

  22. Oh EA, Kim MS, Chi WJ, Kim JH, Hong SK (2007) Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsin. FEMS Microbiol Lett 276:75–82

    Article  PubMed  CAS  Google Scholar 

  23. Ohnishi Y, Ishikawa J, Hara H, Suzuki H, Ikenoya M, Ikeda H, Yamashita A, Hattori M, Horinouchi S (2008) Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J Bacteriol 190:4050–4060

    Article  PubMed  CAS  Google Scholar 

  24. Olafson RW, Jurasek L, Carpenter MR, Smillie LB (1975) Amino acid sequence of Streptomyces griseus trypsin. Cyanogen bromide fragments and complete sequence. Biochemistry 14:1168–1177

    Article  PubMed  CAS  Google Scholar 

  25. Olafson RW, Smillie LB (1975) Enzymic and physicochemical properties of Streptomyces griseus trypsin. Biochemistry 14:1161–1167

    Article  PubMed  CAS  Google Scholar 

  26. Read RJ, James MN (1988) Refined crystal structure of Streptomyces griseus trypsin at 1.7 Å resolution. J Mol Biol 200:523–551

    Article  PubMed  CAS  Google Scholar 

  27. Salamin K, Sriranganadane D, Lechenne B, Jousson O, Monod M (2010) Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71. Appl Environ Microbiol 76:4269–4276

    Article  PubMed  CAS  Google Scholar 

  28. Sidhu SS, Kalmar GB, Willis LG, Borgford TJ (1995) Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes. J Biol Chem 270:7594–7600

    Article  PubMed  CAS  Google Scholar 

  29. Sinha J, Plantz BA, Inan M, Meagher MM (2005) Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau. Biotechnol Bioeng 89:102–112

    Article  PubMed  CAS  Google Scholar 

  30. Stroud RM, Kossiakoff AA, Chambers JL (1977) Mechanisms of zymogen activation. Annu Rev Biophys Bioeng 6:177–193

    Article  PubMed  CAS  Google Scholar 

  31. Trop M, Birk Y (1968) The trypsin-like enzyme from Streptomyces griseus (pronase). Biochem J 109:475–476

    PubMed  CAS  Google Scholar 

  32. Trop M, Birk Y (1970) The specificity of proteinases from Streptomyces griseus (pronase). Biochem J 116:19–25

    PubMed  CAS  Google Scholar 

  33. Vasquez JR, Evnin LB, Higaki JN, Craik CS (1989) An expression system for trypsin. J Cell Biochem 39:265–276

    Article  PubMed  CAS  Google Scholar 

  34. Villatte F, Hussein AS, Bachmann TT, Schmid RD (2001) Expression level of heterologous proteins in Pichia pastoris is influenced by flask design. Appl Microbiol Biot 55:463–465

    Article  CAS  Google Scholar 

  35. Walsh KA (1970) Trypsinogens and trypsins of various species. In: Perlmann G, Lorand L (eds) Methods of enzymology. Academic, New York, pp 41–63

  36. Wang Z, Wang Y, Zhang D, Li J, Hua Z, Du G, Chen J (2010) Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Bioresour Technol 101:1318–1323

    Article  PubMed  CAS  Google Scholar 

  37. Yao LY, Man CX, Zhao F, Qu XG, Han XY, Sun DQ, Jiang YJ (2010) Expression of bovine trypsin in Lactococcus lactis. Int Dairy J 20:806–809

    Article  CAS  Google Scholar 

  38. Yee L, Blanch HW (1993) Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli. Biotechnol Bioeng 41:781–790

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Professor Byong Lee at Jiangnan University for his discussion and revision. We also thank Professor Jian Jin and Ph.D. candidate Ying Hou at the School of Medicine and Pharmaceutics of Jiangnan University for their support with the mammalian cell digestions. This work was financially supported by the National High Technology Research and Development Program of China (863 Program, 2011AA100905), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1135), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Zhenmin Ling, Tengbo Ma & Zhen Kang

  2. School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Zhenmin Ling, Tengbo Ma, Jianghua Li, Guocheng Du, Zhen Kang & Jian Chen

  3. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China

    Jianghua Li & Jian Chen

  4. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Guocheng Du

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  1. Zhenmin Ling
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Correspondence to Zhen Kang or Jian Chen.

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Ling, Z., Ma, T., Li, J. et al. Functional expression of trypsin from Streptomyces griseus by Pichia pastoris . J Ind Microbiol Biotechnol 39, 1651–1662 (2012). https://doi.org/10.1007/s10295-012-1172-3

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  • DOI: https://doi.org/10.1007/s10295-012-1172-3

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