Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
권호 표지
DOI QR코드

DOI QR Code

Expression, Purification, and Characteristic of Tibetan Sheep Breast Lysozyme Using Pichia pastoris Expression System

  • Li, Jianbo (Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation) ;
  • Jiang, Mingfeng (Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation) ;
  • Wang, Yong (Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation)
  • Received : 2013.08.23
  • Accepted : 2013.11.17
  • Published : 2014.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and $45^{\circ}C$. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.

Keywords

References

  1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  2. Davies, R. C., A. Neuberger, and B. M. Wilson. 1969. The dependence of lysozyme activity on pH and ionic strength. Biochem. Biophy. Acta Enzymol. 178:294-305. https://doi.org/10.1016/0005-2744(69)90397-0
  3. Digan, M. E., S. V. Lair, R. A. Brierley, R. S. Siegel, M. E. Williams, S. B. Ellis, P. A. Kellaris, S. A. Provow, W. S. Craig G. Velicelebi, M. M. Harpold, and G. P. Thill. 1989. Continuous production of a novel lysozyme via secretion from the yeast, Pichia pastoris. Nat. Biotechnol. 7:160-164. https://doi.org/10.1038/nbt0289-160
  4. Ferrari, R., C. Callerio, and G. Podio. 1959. Antiviral activity of lysozyme. Nature (Paris) 183:548.
  5. Hughey, V. L. and E. A. Johnson. 1987. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease. Appl. Environ. Microbiol. 53:2165-2170.
  6. Irwin, D. M. and A. C. Wilson. 1990. Concerted evolution of ruminant stomach lysozymes. Characterization of lysozyme cDNA clones from sheep and deer. J. Biol. Chem. 265:4944-4952.
  7. Iwata, T., R. Tanaka, M. Suetsugu, M. Ishibashi, H. Tokunaga, M. Kikuchi, and M. Tokunaga. 2004. Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis. Biotechnol. Lett. 26:1803-1808. https://doi.org/10.1007/s10529-004-4614-9
  8. Kirby, A. J. 2001. The lysozyme mechanism sorted - after 50 years. Nat. Struct. Biol. 8:737-739. https://doi.org/10.1038/nsb0901-737
  9. Leippe, M. 1999. Antimicrobial and cytolytic polypeptides of amoeboid protozoa-effector molecules of primitive phagocytes. Dev. Comp. Immunol. 23:267-279. https://doi.org/10.1016/S0145-305X(99)00010-5
  10. Maurel, P. and P. Douzou. 1976. Catalytic implications of electrostatic potentials: The lytic activity of lysozyme as a model. J. Mol. Biol. 102:253-264. https://doi.org/10.1016/S0022-2836(76)80052-6
  11. Muraki, M., M. Morikawa, Y. Jigami, and H. Tanaka. 1988. Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge. Protein Eng. 2:49-54. https://doi.org/10.1093/protein/2.1.49
  12. O'Fagain, C., P. M. Cummins, and B. F. O'Connor. 2011. Gel-filtration chromatography. Protein Chromatography. Humana Press pp. 25-33.
  13. Ogundele, M. O. 1998. A novel anti-inflammatory activity of lysozyme: modulation of serum complement activation. Mediat. Inflamm. 7:363-365. https://doi.org/10.1080/09629359890893
  14. Parry, R. M., R. C. Chandan, and K. M. Shahani. 1965. A rapid and sensitive assay of muramidase. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, NY). Royal Soc. Med. 119:384-386.
  15. Peeters, T. L., Y. R. Depraetere, and G. R. Vantrappen. 1978. Radioimmunoassay for urinary lysozyme in human serum from leukemic patients. Clin. Chem. 24:2155-2157.
  16. Prager, E. M. and P. Jolies. 1995. Animal lysozymes c and g: an overview in lysozymes: Model enzymes in biochemistry and biology. Birkhauser Basel. pp. 9-31.
  17. Ranieri, M. L., J. R. Huck, M. Sonnen, D. M. Barbano, and K. J. Boor. 2009. High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk. J. Dairy Sci. 92:4823-4832. https://doi.org/10.3168/jds.2009-2144
  18. Raynal-Ljutovac, K., G. Lagriffoul, P. Paccard, I. Guillet, and Y. Chilliard. 2008. Composition of goat and sheep milk products: An update. Small. Rumin. Res. 79:57-72. https://doi.org/10.1016/j.smallrumres.2008.07.009
  19. Shoo, N. R., P. Kumar, B. Bhush, T. K. Bhattacharya, A, Sharma, S. Dayal, P. K. Pankaj, and M. Sahoo. 2010. PCR-SSCP of serum lysozyme gene (Exon-III) in riverine buffalo and its association with lysozyme activity and somatic cell count. Asian-Aust. J. Anim. 23:993-999. https://doi.org/10.5713/ajas.2010.90323
  20. Saint-Blancard, J., P. Chuzel, Y. Mathieu, J. Perrot, and P. Jolles. 1970. Influence of pH and ionic strength on the lysis of Micrococcus lysodeikticus cells by six human and four avian lysozymes. Biochem. Biophys. Acta Enzymol. 220:300-306. https://doi.org/10.1016/0005-2744(70)90014-8
  21. Salton, M. R. and J. M. Ghuysen. 1959. The structure of di- and tetrasaccharides released from cell walls by lysozyme and Streptomyces F1 enzyme and the beta (1 to 4) N-acetylhexos-aminidase activity of these enzymes. Biochim. Biophys. Acta. Dec. 36:552-554. https://doi.org/10.1016/0006-3002(59)90205-7
  22. Sava, G., A. Benetti, V. Ceschia, and S. Pacor. 1989. Lysozyme and cancer: role of exogenous lysozyme as anticancer agent (review). Anticancer. Res. 9:583.
  23. Su, C. K. and B. H. Chiang. 2006. Partitioning and purification of lysozyme from chicken egg white using aqueous two-phase system. Proc. Biochem. 41:257-263. https://doi.org/10.1016/j.procbio.2005.06.026
  24. Vasstrand, E. N. and H. B. Jensen. 1980. Affinity chromatography of human saliva lysozyme and effect of pH and ionic strength on lytic activity. Eur. J. Oral Sci. 88:219-228. https://doi.org/10.1111/j.1600-0722.1980.tb01218.x
  25. Wilken, L. R. and Z. L. Nikolov. 2006. Factors influencing recombinant human lysozyme extraction and cation exchange adsorption. Biotechnol. Progr. 22:745-752. https://doi.org/10.1021/bp0600536
  26. Xue, Q. G., K. L. Schey, A. K. Volety, F. L. Chu, and J. F. La Peyre. 2004. Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 139:11-25. https://doi.org/10.1016/j.cbpc.2004.05.011
  27. Yang, B., J. Wang, B. Tang, Y. Liu, C. Guo, P. Yang, T. Yu, R. Li, J. Zhao, L. Zhang, Y. Dai, and N. Li. 2011. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle. Plos One 6(3):e17593. https://doi.org/10.1371/journal.pone.0017593

Cited by

  1. Effects of ink characteristics and piezo-electric inkjetting parameters on lysozyme activity vol.9, pp.None, 2014, https://doi.org/10.1038/s41598-019-54723-9