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Expression, Purification, and Antibacterial Activity of Bovine Lactoferrampin–Lactoferricin in Pichia pastoris

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Abstract

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N1 domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA–LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA–LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA–LFA–LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA–LFC peptide was purified via two-step column chromatography and identified by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA–LFC. The fermentation supernate and the purified LFA–LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA–LFC in P. pastoris and may have potential practical applications in microbial peptide production.

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References

  1. Kuwata, H., Yip, T.-T., Yip, C. L., Tomita, M., & Hutchens, T. W. (1998). Biochemical and Biophysical Research Communications, 245, 764–773.

    Article  CAS  Google Scholar 

  2. Masschalck, B., Van Houdt, R., & Michiels, C. W. (2001). International Journal of Food Microbiology, 64, 325–332.

    Article  CAS  Google Scholar 

  3. Van der Kraan, M. I. A., Groenink, J., Nazmi, K., Veerman, E. C. I., Bolscher, J. G. M., & Nieuw Amerongen, A. V. (2004). Peptides, 25, 177–183.

    Article  Google Scholar 

  4. Haney, E. F., Nazmi, K., Lau, F., Bolscher, J. G. M., & Vogel, H. J. (2009). Biochimie, 91, 141–154.

    Article  CAS  Google Scholar 

  5. Haney, E. F., Lau, F., & Vogel, H. J. (2007). Biochimica et Biophysica Acta (BBA)-Biomembranes, 1768, 2355–2364.

    Article  CAS  Google Scholar 

  6. Farnaud, S., Spiller, C., Moriarty, L. C., Patel, A., Gant, V., Odell, E. W., & Evans, R. W. (2004). FEMS Microbiology Letters, 233, 193–199.

    Article  CAS  Google Scholar 

  7. Ulvatne, H., Samuelsen, Ø., Haukland, H. H., Kräer, M., & Vorland, L. H. (2004). FEMS Microbiology Letters, 237, 377–384.

    CAS  Google Scholar 

  8. Zweytick, D., Tumer, S., Blondelle, S. E., & Lohner, K. (2008). Biochemical and Biophysical Research Communications, 369, 395–400.

    Article  CAS  Google Scholar 

  9. Chen, G. H., Chen, W. M., Huang, G. T., Chen, Y. W., & Jiang, S. T. (2009). Journal of Agricultural and Food Chemistry, 57(20), 9509–9515.

    Article  CAS  Google Scholar 

  10. Ellis, R. E., & Greathouse, D. (2009). Biophysical Journal, 96, 609a–609a.

    Article  Google Scholar 

  11. Bolscher, J. G. M., Adão, R., Nazmi, K., Van den Keybus, P. A. M., van 't Hof, W., Nieuw Amerongen, A. V., Bastos, M., & Veerman, E. C. I. (2009). Biochimie, 91, 123–132.

    Article  CAS  Google Scholar 

  12. Marr, A. K., Jenssen, H., Moniri, M. R., Hancock, R. E. W., & Pant, N. (2009). Biochimie, 91, 160–164.

    Article  CAS  Google Scholar 

  13. Jenssen, H., Sandvik, K., Andersen, J. H., Hancock, R. E. W., & Gutteberg, T. J. (2008). Antiviral Research, 79, 192–198.

    Article  CAS  Google Scholar 

  14. Mistry, N., Drobni, P., Näslund, J., Sunkari, V. G., Jenssen, H., & Evander, M. (2007). Antiviral Research, 75, 258–265.

    Article  CAS  Google Scholar 

  15. Jenssen, H., Andersen, J. H., Uhlin-Hansen, L., Gutteberg, T. J., & Rekdal, Ø. (2004). Antiviral Research, 61, 101–109.

    Article  CAS  Google Scholar 

  16. Freiburghaus, C., Månsson, H. L., Oredsson, S. M., & Paulsson, M. (2008). European Journal of Cancer Supplements, 6, 150–150.

    Article  Google Scholar 

  17. Regina, A., Kamran, N., Jan, G. M., & Margarida, B. (2011). Journal of Pharmacy and Bioallied Sciences, 3, 60–69.

    Article  Google Scholar 

  18. Xu, G., Xiong, W., Hu, Q., Zuo, P., Shao, B., Lan, F., Lu, X., Xu, Y., & Xiong, S. (2010). Journal of Applied Microbiology, 109, 1311–1318.

    Article  CAS  Google Scholar 

  19. Zhou, Q., Li, M., & Xi, T. (2009). Current Microbiology, 58, 384–388.

    Article  CAS  Google Scholar 

  20. Li, Z., Moy, A., Sohal, K., Dam, C., Kuo, P., Whittaker, J., Whittaker, M., Duzgunes, N., Konopka, K., Franz, A. H., Lin-Cereghino, J., & Lin-Cereghino, G. P. (2009). Protein Expression and Purification, 67, 175–181.

    Article  CAS  Google Scholar 

  21. Wang, A., Wang, S., Shen, M., Chen, F., Zou, Z., Ran, X., Cheng, T., Su, Y., & Wang, J. (2009). Applied Microbiology and Biotechnology, 84, 877–884.

    Article  CAS  Google Scholar 

  22. Dong, Z. Y., & Zhang, Y. Z. (2006). Biotechnology Letters, 28, 1285–1292.

    Article  CAS  Google Scholar 

  23. Tang, Z., Zhang, Y., Stewart, A. F., Geng, M., Tang, X., Tu, Q., & Yin, Y. (2010). Protein Expression and Purification, 73, 132–139.

    Article  CAS  Google Scholar 

  24. Liu, Z. M., Lu, Z. X., & Lv, F. X. (2006). Process Biochemistry, 41, 975–979.

    Article  CAS  Google Scholar 

  25. Schägger, H. (2006). Tricine–SDS–PAGE. Nature Protocols, 1, 16–22.

    Article  Google Scholar 

  26. Schagger, H., & Von, J. G. (1987). Analytical Biochemistry, 166, 368–379.

    Article  CAS  Google Scholar 

  27. Yang, Y., Tian, Z., Teng, D., Zhang, J., Wang, J., & Wang, J. (2009). Biotechnology, 139, 326–331.

    CAS  Google Scholar 

  28. Martin, M., Gopinath, K., Mukesh, P., Jan, A., & Artur, S. (2011). PLoS One, 6(1), e16400.

    Article  Google Scholar 

  29. Heiman, M., Engel, A., & Walter, P. (2007). The Journal of Cell Biology, 176, 209–222.

    Article  CAS  Google Scholar 

  30. Alison, B., Charles, B., & Robert, S. F. (1998). Proceedings of the National Academy of Sciences, 95, 10384–10389.

    Article  Google Scholar 

  31. Clare, J., Romanos, M., Rayment, F., Rowedder, J., Smith, M., Payne, M., Sreekrishna, K., & Henwood, C. (1991). Gene, 105, 205–212.

    Article  CAS  Google Scholar 

  32. Zhang, J., Zhang, Sq, Wu, X., Chen, Yq, & Diao, Zy. (2006). Process Biochemistry, 41, 251–256.

    Article  CAS  Google Scholar 

  33. Matsumoto, H., Hashimoto, K., Takatsu, Z., Shimamura, S., & Tomita, M. (1996). Journal of Infection and Chemotherapy, 1, 185–189.

    Article  Google Scholar 

Download references

Acknowledgments

This research was supported by grants from the Chinese Academy of Sciences and Knowledge Innovation Project (KZCX2-EW-412KSCX2-EW-B-10;KZCX2-EW-412), NSFC (31110103909; 30700581), Hunan Provincial Natural Science Foundation of China (10JJ4013), Program for Doctor Fund of Southwest University (SWU111002), the Program for the Latest Research Field of Youth Talents in the Chinese Academy of Sciences (ISACX-LYQY-QN-0701), the National “863” Program of China (2008AA10Z316), Program for overall strategy cooperation between Guandong province and Chinese Acadamy of Science (2010B090300038), and the CAS/SAFEA International Partnership Program for Creative Research Teams.

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Authors and Affiliations

  1. Research Center for Healthy Breeding of Livestock and Poultry, Institute of Subtropical Agriculture, CAS, Changsha, Hunan, 410125, China

    Xiang-Shan Tang, Sheng-Ping Wang, Ze-Meng Feng, Dong Zhou, Tie-Jun Li & Yu-Long Yin

  2. Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, CAS, Changsha, Hunan, 410125, China

    Xiang-Shan Tang, Sheng-Ping Wang, Ze-Meng Feng, Dong Zhou, Tie-Jun Li & Yu-Long Yin

  3. College of Animal Science and Technology, Southwest University, Chongqing, 400715, China

    Zhi-Ru Tang

  4. Graduate School of Chinese Academy of Sciences, Beijing, 100049, China

    Xiang-Shan Tang, Sheng-Ping Wang, Ze-Meng Feng & Dong Zhou

  5. Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, 410125, China

    Yu-Long Yin

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  1. Xiang-Shan Tang
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  2. Zhi-Ru Tang
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Correspondence to Yu-Long Yin.

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Tang, XS., Tang, ZR., Wang, SP. et al. Expression, Purification, and Antibacterial Activity of Bovine Lactoferrampin–Lactoferricin in Pichia pastoris . Appl Biochem Biotechnol 166, 640–651 (2012). https://doi.org/10.1007/s12010-011-9455-0

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  • DOI: https://doi.org/10.1007/s12010-011-9455-0

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