Expression and Purification of the Main Component Contained in Camel Milk and Its Antimicrobial Activities Against Bacterial Plant Pathogens

  • Abbas Tanhaeian
  • Farajollah Shahriari Ahmadi
  • Mohammad Hadi Sekhavati
  • Mojtaba Mamarabadi
Article
  • 54 Downloads

Abstract

Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 μg/ml for different bacterial isolates.

Keywords

Lactoferricin Lactoframpin HEK293 Recombinant peptide Plant bacterial pathogens 

Notes

Acknowledgments

We thank Dr. Hesamoddin Shahriari for editing the manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Parc AL, Karav S, Rouquié C, Maga EA, Bunyatratchata A, Barile D (2017) Characterization of recombinant human lactoferrin N-glycans expressed in the milk of transgenic cows. PLoS One 12(2):e0171477.  https://doi.org/10.1371/journal.pone.0171477 CrossRefGoogle Scholar
  2. 2.
    Khaldi N, Shields DC (2001) Shift in the isoelectric-point of milk proteins as a consequence of adaptive divergence between the milks of mammalian species. Biol Direct 6(1):40.  https://doi.org/10.1186/1745-6150-6-40 CrossRefGoogle Scholar
  3. 3.
    Gupta C, Prakash D (2017) Therapeutic potential of milk whey. Beverages 3(3):31.  https://doi.org/10.3390/beverages3030031 CrossRefGoogle Scholar
  4. 4.
    Rachman AB, Maheswari RR, Bachroem MS (2015) Composition and isolation of lactoferrin from colostrum and milk of various goat breeds. Procedia Food Sci 3:200–210.  https://doi.org/10.1016/j.profoo.2015.01.022 CrossRefGoogle Scholar
  5. 5.
    Legrand D, Pierce A, Elass E, Carpentier M, Mariller C, Mazurier J (2008) Lactoferrin structure and functions. In: Bösze Z (ed) Bioactive components of milk. Advances in Experimental Medicine and Biology, vol 606. Springer, New York, pp 163–194.  https://doi.org/10.1007/978-0-387-74087-4_6. CrossRefGoogle Scholar
  6. 6.
    Actor JK, Hwang SA, Kruzel ML (2009) Lactoferrin as a natural immune modulator. Curr Pharm Des 15(17):1956–1973.  https://doi.org/10.2174/138161209788453202 CrossRefGoogle Scholar
  7. 7.
    Artym J (2010) The role of lactoferrin in the iron metabolism. Part II. Antimicrobial and antiinflammatory effect of lactoferrin by chelation of iron. Postepy Hig Med Dosw 64:604–616Google Scholar
  8. 8.
    Cassat JE, Skaar EP (2013) Iron in infection and immunity. Cell Host Microbe 13(5):509–519.  https://doi.org/10.1016/j.chom.2013.04.010 CrossRefGoogle Scholar
  9. 9.
    González-Chávez SA, Arévalo-Gallegos S, Rascón-Cruz Q (2009) Lactoferrin: structure, function and applications. J Antimicrob Agents 33(4):301–3e1.  https://doi.org/10.1016/j.ijantimicag.2008.07.020 CrossRefGoogle Scholar
  10. 10.
    Tomita M, Wakabayashi H, Shin K, Yamauchi K, Yaeshima T, Iwatsuki K (2009) Twenty-five years of research on bovine lactoferrin applications. Biochimie 91(1):52–57.  https://doi.org/10.1016/j.biochi.2008.05.021 CrossRefGoogle Scholar
  11. 11.
    Bruni N, Capucchio MT, Biasibetti E, Pessione E, Cirrincione S, Giraudo L, Corona A, Dosio F (2016) Antimicrobial activity of lactoferrin-related peptides and applications in human and veterinary medicine. Molecules 21(6):752.  https://doi.org/10.3390/molecules21060752 CrossRefGoogle Scholar
  12. 12.
    Legrand D, Elass E, Carpentier M, Mazurier J (2005) Lactoferrin: a modulator of immune and inflammatory responses. Cell Mol Life Sci 62(22):2549–2559.  https://doi.org/10.1007/s00018-005-5370-2 CrossRefGoogle Scholar
  13. 13.
    Sijbrandij T, Ligtenberg AJ, Nazmi K, Veerman EC, Bolscher JG, Bikker FJ (2017) Effects of lactoferrin derived peptides on simulants of biological warfare agents. World J Microbiol Biotechnol 33(1):3.  https://doi.org/10.1007/s11274-016-2171-8 CrossRefGoogle Scholar
  14. 14.
    Sinha M, Kaushik S, Kaur P, Sharma S, Singh TP (2013) Antimicrobial lactoferrin peptides: the hidden players in the protective function of a multifunctional protein. Int J Pept 390230, 12 pages:1–12.  https://doi.org/10.1155/2013/390230 CrossRefGoogle Scholar
  15. 15.
    Bolscher JG, Adão R, Nazmi K, van den Keybus PA, van’t Hof W, Amerongen AV, Bastos M, Veerman EC (2009) Bactericidal activity of LFchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides. Biochimie 91(1):123–132.  https://doi.org/10.1016/j.biochi.2008.05.019 CrossRefGoogle Scholar
  16. 16.
    Tang XS, Tang ZR, Wang SP, Feng ZM, Zhou D, Li TJ, Yin YL (2012) Expression, purification, and antibacterial activity of bovine lactoferrampin–lactoferricin in Pichia pastoris. Appl Biochem Biotechnol 166(3):640–651.  https://doi.org/10.1007/s12010-011-9455-0 CrossRefGoogle Scholar
  17. 17.
    Dheeb BI, Al-Mudallal NH, Salman ZA, Ali M, Nouri MA, Hussain HT, Abdulredha SS (2015) The inhibitory effects of human, camel and cow’s milk against some pathogenic fungi in Iraq. Jordan J Biol Sci 8(2):89–93.  https://doi.org/10.12816/0027553 CrossRefGoogle Scholar
  18. 18.
    Lin YC, Boone M, Meuris L, Lemmens I, Van Roy N, Soete A, Reumers J, Moisse M, Plaisance S, Drmanac R, Chen J (2014) Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat Commun 5:4767.  https://doi.org/10.1038/ncomms5767 CrossRefGoogle Scholar
  19. 19.
    Thomas P, Smart TG (2005) HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 51(3):187–200.  https://doi.org/10.1016/j.vascn.2004.08.014 CrossRefGoogle Scholar
  20. 20.
    Chen GH, Chen WM, Huang GT, Chen YW, Jiang ST (2009) Expression of recombinant antibacterial lactoferricin-related peptides from Pichia pastoris expression system. J Agric Food Chem 57(20):9509–9515.  https://doi.org/10.1021/jf902611h CrossRefGoogle Scholar
  21. 21.
    Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31(13):3381–3385.  https://doi.org/10.1093/nar/gkg520 CrossRefGoogle Scholar
  22. 22.
    Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ (2005) GROMACS: fast, flexible, and free. J Comput Chem 26(16):1701–1718.  https://doi.org/10.1002/jcc.20291 CrossRefGoogle Scholar
  23. 23.
    Jordan M, Schallhorn A, Wurm FM (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4):596–601.  https://doi.org/10.1093/nar/24.4.596 CrossRefGoogle Scholar
  24. 24.
    Schägger H, Aquila H, Von Jagow G (1988) Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis. Anal Biochem 173(1):201–205.  https://doi.org/10.1016/0003-2697(88)90179-0 CrossRefGoogle Scholar
  25. 25.
    Vipra A, Desai SN, Junjappa RP, Roy P, Poonacha N, Ravinder P, Sriram B, Padmanabhan S (2013) Determining the minimum inhibitory concentration of bacteriophages: potential advantages. Adv Microbiol 3(02):181–190.  https://doi.org/10.4236/aim.2013.32028 CrossRefGoogle Scholar
  26. 26.
    Chahardooli M, Niazi A, Aram F, Sohrabi SM (2016) Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification. J Sci Food Agric 96(2):569–575.  https://doi.org/10.1002/jsfa.7125 CrossRefGoogle Scholar
  27. 27.
    Xu XW, Pei SJ, Miao XR, Yu WF (2009) Human signal peptide had advantage over mouse in secretory expression. Histochem Cell Biol 132(2):239–246.  https://doi.org/10.1007/s00418-009-0602-4 CrossRefGoogle Scholar
  28. 28.
    Agrios GN (2005) Plant pathology, 5th edn. Academic Press, San DiegoGoogle Scholar
  29. 29.
    Kovalskaya N, Hammond RW (2009) Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins. Protein Expr Purif 63(1):12–17.  https://doi.org/10.1016/j.pep.2008.08.013 CrossRefGoogle Scholar
  30. 30.
    Chen GH, Yin LJ, Chiang IH, Jiang ST (2007) Expression and purification of goat lactoferrin from Pichia pastoris expression system. J Food Sci 72:67–71.  https://doi.org/10.1111/j.1750-3841.2007.00281.x CrossRefGoogle Scholar
  31. 31.
    Lai Y, Gallo RL (2009) AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol 30(3):131–141.  https://doi.org/10.1016/j.it.2008.12.003 CrossRefGoogle Scholar
  32. 32.
    Strøm MB, Svendsen JS, Rekdal Ø (2000) Antibacterial activity of 15-residue lactoferricin derivatives. Chem Biol Drug Des 56(5):265–274.  https://doi.org/10.1034/j.1399-3011.2000.00770.x Google Scholar
  33. 33.
    Chan DI, Prenner EJ, Vogel HJ (2006) Tryptophan-and arginine-rich antimicrobial peptides: structures and mechanisms of action. BBA Biomembranes 1758(9):1184–1202.  https://doi.org/10.1016/j.bbamem.2006.04.006 CrossRefGoogle Scholar
  34. 34.
    Phoenix DA, Dennison SR, Harris F (2013) Models for the membrane interactions of antimicrobial peptides. In: Phoenix DA, Dennison SR, Harris F (eds) Antimicrobial peptides. Wiley, Weinheim, pp 145–180.  https://doi.org/10.1002/9783527652853.index CrossRefGoogle Scholar
  35. 35.
    Rossi P, Giansanti F, Boffi A, Ajello M, Valenti P, Chiancone E, Antonini G (2002) Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide. Biochem Cell Biol 80(1):41–48.  https://doi.org/10.1139/o01-209 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Abbas Tanhaeian
    • 1
  • Farajollah Shahriari Ahmadi
    • 1
  • Mohammad Hadi Sekhavati
    • 2
  • Mojtaba Mamarabadi
    • 3
  1. 1.Department of Biotechnology and Plant Breeding, Faculty of AgricultureFerdowsi University of MashhadMashahadIran
  2. 2.Department of Animal Sciences, Faculty of AgricultureFerdowsi University of MashhadMashahadIran
  3. 3.Department of Plant Protection, Faculty of AgricultureFerdowsi University of MashhadMashahadIran

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